Cardiovascular Research 1992;26:897-905
897
Interdependence of intracellular taurine and sodium in guinea pig heart Moh'd-Saadeh Suleiman, Glenn C Rodrigo, and Reg A Chapman
T
he amino acid taurine (2-amino ethanesulphonic acid), which is not incorporated into proteins and is very slowly metabolised, constitutes almost half of the amino acids free in the sarcoplasm of mammalian heart cells. Although taurine remains without a well defined physiological role in the heart,' it is clearly implicated in the maintenance of normal cardiac cellular function, as illustrated by the fact that its depletion is associated with the development of cardiomyopathy' and that its presence affects the conse uences of ischaemia, hypoxia, and the calcium paradox.4 3 Recently there has been some debate concerning the origins of the calcium paradox in cardiac One of the key questions stems from the discrepancy between laboratories as to whether or not isolated m tes are damaged by Ca depletion followed by repletion! 'The Ca influx on Ca repletion, and the number of cultured (mouse) myocytes damaged, shows a sigmoidal dependence on the pCa of the solution used during Ca dep1eti0n.l~A similar relationship is seen in guinea pig ventricular myocytes isolated in normal v r o d e solution. However, the relationship of pCa versus the percentage of cells unable to exclude the dye Trypan Blue is shifted by 1 pCa unit towards higher pCa values in myocytes isolated in the presence of 40 mM taurine, an effect that is antagonised if the Ca depletion is
2:
~
done in the presence of taurine, p alanine, or haloperidol." These results suggest that the presence of high concentrations of taurine (or other amino acids) seen in the KB or the culture media used in the isolation and/or storage of enzymatically isolated myocytes in some laboratories affects their sensitivity to Ca depletion and Ca repletion, probably by establishing raised intracellular taurine concentrations. Indeed, in the original paper where the use of KB medium was introduced, the importance of taurine was noted.14 The transport of amino acids, including taurine, occurs across many cell membranes and is often accompanied by the movement of Na+.19 20 Should a similar transport system be present in cardiac muscle and be able to reverse, an explanation for many of the effects of taurine and for the difference in the sensitivity of isolated myocytes to damage can be proposed in terms of an action on [Nali. To test this notion, we describe experiments, which show an effect of raised intracellular taurine on a d in isolated myocytes, and the effect on intracellular taurine concentrations of experimental changes that will alter intracellular sodium, measured in perfused guinea pig hearts. Preliminary results of this work have been reported." 22 Methods Male guinea pigs (300-350 g) were killed by spinal dislocation and the
~~
British Heart Foundation Research Group in Cellular Cardiology, Department of Physiology, School of Veterinary Science, University of Bristol, Park Row, Bristol BS1 5LS, United Kingdom: M-S Suleiman, G C Rodrigo, R A Chapman. Correspondence to Professor Chapman.
Downloaded from by guest on October 27, 2014
Objective: The aim was to investigate the effects of raising intracellular taurine on the intracellular sodium activity (am') in isolated guinea pig ventricular myocytes, and the effect of procedures that raise intracellular sodium on taurine concentration in the perfused guinea pig ventricular tissue. Methods: Taurine was introduced into the sarcoplasm of isolated ventricular myocytes, either during cell isolation or by diffusion from a penetrating micropipette, and the effect on aN; was measured using an ion sensitive microelectrode. Guinea pig hearts, mounted on a Langendorff apparatus, were perfused with a variety of physiological media and the level of taurine in the ventricles determined using high pressure liquid chromatography. Results: An increase in intracellular taurine caused by its presence during cell isolation or by diffusion from a micropipette significantly reduced the aNal of isolated myocytes at rest, during perfusion with Ca depleted solutions, or on inhibition of the Na pump. In the guinea pig ventricles, taurine at 13.0(SEM 0.6) mmoLkg-' wet weight comprised up to 45% of the free amino acids; since plasma taurine was 64(13) pmol.litre-', this means that in vivo a large outwardly directed gradient for taurine exists (equivalent to a free energy of 13.7 KJemol-I). Upon perfusion with Ca,Mg free Tyrode solution (which raises intracellular sodium markedly), a time dependent loss of taurine occurred. Both the rate of loss and the total amount lost were increased when the Na pump was also inhibited. This loss of tissue taurine was not due to release from dead or lysed cells, as it was antagonised by procedures known to reduce the rise of aNaiduring Ca depletion, was inhibited by p alanine (an inhibitor of taurine transport), and the fall in tissue taurine was not correlated with the appearance of lactate dehydrogenase in the effluent. Conclusions: The data from isolated myocytes and perfused guinea pig h e q s were consistent with the presence of a Ndtaurine symport which is activated to cause efflux of Na and taurine when either rise above their physiological level. Cardiovascular Research 1992;26:897-905
898
Suleiman, Rodrigo, Chapman
hearts quickly removed and mounted on a conventional Langendorff apparatus. Hearts were perfused with oxygenated normal Tyrode solution, composition (mmol4tre-I): NaCl 135, KCl 5.4, CaC12 2, MgClz 1, Na pyruvate 5, NaH2P04 0.33, glucose 10, HEPES-NaOH 10, at pH 7.3 and a constant flow of 8 m1.min-I at 35°C. Preparation of isolated guinea pig ventricular myocytes The method for isola@g myocytes is essentially the same as described by Mitra and Morad. The heart was perfused with oxygenated Tyrode solution for 5 min to clear the blood and for a further 5 min in nominally Ca free Tyrode. The enzyme solution (1 m g d collagenase 1 and 0.3 m g d protease type XIV with 1% bovine serum albumin, fraction V, Sigma, St Louis, MO, USA) was perfused until myocytes were detected in the effluent. The heart was then perfused for 5 min with normal Tyrode solution and then removed, cut into small pieces, and gently agitated for 10 min in 5 ml normal Tyrode in a shaking water bath at 35°C. For myocytes isolated in Tyrode plus 50 mM taurine or into KB medium (composition described el~ewhere'~),the taurine containing Tyrode or the KE3 medium replaced normal Tyrode in the final perfusion and during the mechanical agitation. The yield of rod shaped myocytes isolated into normal Tyrode varied between 50 and 80%. and when Tyrode with added taurine or KB media was used, the yield was 70-90% (as determined by counting 100-200 myocytes under the microscope). The myocytes, irrespective of how they were prepared, were washed several times in normal Tyrode by centrifugation at 500 g for 2 min and stored in normal Tyrode at room temperature (18-24°C). The data reported below were obtained from isolations using the same batch of enzymes.
were digitally recorded and stored on disc using the MacLab system (WPI) together with a Macintosh Plus or SE130 microcomputer. Measurement of tissue taurine levels in perfused guinea pig hearts Upon the completion of perfusion by the Langendorff method, the ventricles were removed, blotted dry using filter paper, weighed (procedure lasting 30-40 s), and homogenised for 30 s in 5 ml ice cold HPLC water, the homogenate being then further diluted into 50 ml HzO. Aliquots of the homogenate (0.2 ml) were added to Millipore ultrafiltration units and filtered by combined filtration centrifugation at 15800 g and 20°C. using an Eppendorf refrigerated centrifuge 5402. The filtrate was then vacuum dried and the phenylisothiocarbamyl derivitised amino acids" were separated by HPLC on a 15 cm X 4.6 cm Spherisorb 3 umODS2 column using two Waters 510 delivery systems. The solvents used were (a) 0.14 M Na acetate + 850 pllitretriethylamine, pH 5.6, and (b) 60% acetonitrile; with a gradient of O%b for 1.2 min; 0-?2%b for 10.8 min (convex curve); and 100%b for 4 min, at 0.8 mhmin- . Determination of lactate dehydrogenase activity in the peifusate Lactate dehydrogenase activity was determined in 50 p,I aliquots of the effluent taken at regular intervals. A commercially available kit (Boehringer Mannheim GmbH, No 124907) was used and the values were expressed as Ulitre-'. To correct for heart weights, the final value of each measurement is expressed as U.litre-'.rng- dry weight. Total protein level, measured by absorption at 280 nm, and creatine kinase were determined together with lactate dehydrogenase. The time course for each was virtually identical and therefore only data for lactate dehydrogenase are shown. C~
Statistics Data are expressed as mean(SEM) and n refers to the number of experiments. Except for data presented in table 11, where a paired t test was used, statistical analysis at p
897
Interdependence of intracellular taurine and sodium in guinea pig heart Moh'd-Saadeh Suleiman, Glenn C Rodrigo, and Reg A Chapman
T
he amino acid taurine (2-amino ethanesulphonic acid), which is not incorporated into proteins and is very slowly metabolised, constitutes almost half of the amino acids free in the sarcoplasm of mammalian heart cells. Although taurine remains without a well defined physiological role in the heart,' it is clearly implicated in the maintenance of normal cardiac cellular function, as illustrated by the fact that its depletion is associated with the development of cardiomyopathy' and that its presence affects the conse uences of ischaemia, hypoxia, and the calcium paradox.4 3 Recently there has been some debate concerning the origins of the calcium paradox in cardiac One of the key questions stems from the discrepancy between laboratories as to whether or not isolated m tes are damaged by Ca depletion followed by repletion! 'The Ca influx on Ca repletion, and the number of cultured (mouse) myocytes damaged, shows a sigmoidal dependence on the pCa of the solution used during Ca dep1eti0n.l~A similar relationship is seen in guinea pig ventricular myocytes isolated in normal v r o d e solution. However, the relationship of pCa versus the percentage of cells unable to exclude the dye Trypan Blue is shifted by 1 pCa unit towards higher pCa values in myocytes isolated in the presence of 40 mM taurine, an effect that is antagonised if the Ca depletion is
2:
~
done in the presence of taurine, p alanine, or haloperidol." These results suggest that the presence of high concentrations of taurine (or other amino acids) seen in the KB or the culture media used in the isolation and/or storage of enzymatically isolated myocytes in some laboratories affects their sensitivity to Ca depletion and Ca repletion, probably by establishing raised intracellular taurine concentrations. Indeed, in the original paper where the use of KB medium was introduced, the importance of taurine was noted.14 The transport of amino acids, including taurine, occurs across many cell membranes and is often accompanied by the movement of Na+.19 20 Should a similar transport system be present in cardiac muscle and be able to reverse, an explanation for many of the effects of taurine and for the difference in the sensitivity of isolated myocytes to damage can be proposed in terms of an action on [Nali. To test this notion, we describe experiments, which show an effect of raised intracellular taurine on a d in isolated myocytes, and the effect on intracellular taurine concentrations of experimental changes that will alter intracellular sodium, measured in perfused guinea pig hearts. Preliminary results of this work have been reported." 22 Methods Male guinea pigs (300-350 g) were killed by spinal dislocation and the
~~
British Heart Foundation Research Group in Cellular Cardiology, Department of Physiology, School of Veterinary Science, University of Bristol, Park Row, Bristol BS1 5LS, United Kingdom: M-S Suleiman, G C Rodrigo, R A Chapman. Correspondence to Professor Chapman.
Downloaded from by guest on October 27, 2014
Objective: The aim was to investigate the effects of raising intracellular taurine on the intracellular sodium activity (am') in isolated guinea pig ventricular myocytes, and the effect of procedures that raise intracellular sodium on taurine concentration in the perfused guinea pig ventricular tissue. Methods: Taurine was introduced into the sarcoplasm of isolated ventricular myocytes, either during cell isolation or by diffusion from a penetrating micropipette, and the effect on aN; was measured using an ion sensitive microelectrode. Guinea pig hearts, mounted on a Langendorff apparatus, were perfused with a variety of physiological media and the level of taurine in the ventricles determined using high pressure liquid chromatography. Results: An increase in intracellular taurine caused by its presence during cell isolation or by diffusion from a micropipette significantly reduced the aNal of isolated myocytes at rest, during perfusion with Ca depleted solutions, or on inhibition of the Na pump. In the guinea pig ventricles, taurine at 13.0(SEM 0.6) mmoLkg-' wet weight comprised up to 45% of the free amino acids; since plasma taurine was 64(13) pmol.litre-', this means that in vivo a large outwardly directed gradient for taurine exists (equivalent to a free energy of 13.7 KJemol-I). Upon perfusion with Ca,Mg free Tyrode solution (which raises intracellular sodium markedly), a time dependent loss of taurine occurred. Both the rate of loss and the total amount lost were increased when the Na pump was also inhibited. This loss of tissue taurine was not due to release from dead or lysed cells, as it was antagonised by procedures known to reduce the rise of aNaiduring Ca depletion, was inhibited by p alanine (an inhibitor of taurine transport), and the fall in tissue taurine was not correlated with the appearance of lactate dehydrogenase in the effluent. Conclusions: The data from isolated myocytes and perfused guinea pig h e q s were consistent with the presence of a Ndtaurine symport which is activated to cause efflux of Na and taurine when either rise above their physiological level. Cardiovascular Research 1992;26:897-905
898
Suleiman, Rodrigo, Chapman
hearts quickly removed and mounted on a conventional Langendorff apparatus. Hearts were perfused with oxygenated normal Tyrode solution, composition (mmol4tre-I): NaCl 135, KCl 5.4, CaC12 2, MgClz 1, Na pyruvate 5, NaH2P04 0.33, glucose 10, HEPES-NaOH 10, at pH 7.3 and a constant flow of 8 m1.min-I at 35°C. Preparation of isolated guinea pig ventricular myocytes The method for isola@g myocytes is essentially the same as described by Mitra and Morad. The heart was perfused with oxygenated Tyrode solution for 5 min to clear the blood and for a further 5 min in nominally Ca free Tyrode. The enzyme solution (1 m g d collagenase 1 and 0.3 m g d protease type XIV with 1% bovine serum albumin, fraction V, Sigma, St Louis, MO, USA) was perfused until myocytes were detected in the effluent. The heart was then perfused for 5 min with normal Tyrode solution and then removed, cut into small pieces, and gently agitated for 10 min in 5 ml normal Tyrode in a shaking water bath at 35°C. For myocytes isolated in Tyrode plus 50 mM taurine or into KB medium (composition described el~ewhere'~),the taurine containing Tyrode or the KE3 medium replaced normal Tyrode in the final perfusion and during the mechanical agitation. The yield of rod shaped myocytes isolated into normal Tyrode varied between 50 and 80%. and when Tyrode with added taurine or KB media was used, the yield was 70-90% (as determined by counting 100-200 myocytes under the microscope). The myocytes, irrespective of how they were prepared, were washed several times in normal Tyrode by centrifugation at 500 g for 2 min and stored in normal Tyrode at room temperature (18-24°C). The data reported below were obtained from isolations using the same batch of enzymes.
were digitally recorded and stored on disc using the MacLab system (WPI) together with a Macintosh Plus or SE130 microcomputer. Measurement of tissue taurine levels in perfused guinea pig hearts Upon the completion of perfusion by the Langendorff method, the ventricles were removed, blotted dry using filter paper, weighed (procedure lasting 30-40 s), and homogenised for 30 s in 5 ml ice cold HPLC water, the homogenate being then further diluted into 50 ml HzO. Aliquots of the homogenate (0.2 ml) were added to Millipore ultrafiltration units and filtered by combined filtration centrifugation at 15800 g and 20°C. using an Eppendorf refrigerated centrifuge 5402. The filtrate was then vacuum dried and the phenylisothiocarbamyl derivitised amino acids" were separated by HPLC on a 15 cm X 4.6 cm Spherisorb 3 umODS2 column using two Waters 510 delivery systems. The solvents used were (a) 0.14 M Na acetate + 850 pllitretriethylamine, pH 5.6, and (b) 60% acetonitrile; with a gradient of O%b for 1.2 min; 0-?2%b for 10.8 min (convex curve); and 100%b for 4 min, at 0.8 mhmin- . Determination of lactate dehydrogenase activity in the peifusate Lactate dehydrogenase activity was determined in 50 p,I aliquots of the effluent taken at regular intervals. A commercially available kit (Boehringer Mannheim GmbH, No 124907) was used and the values were expressed as Ulitre-'. To correct for heart weights, the final value of each measurement is expressed as U.litre-'.rng- dry weight. Total protein level, measured by absorption at 280 nm, and creatine kinase were determined together with lactate dehydrogenase. The time course for each was virtually identical and therefore only data for lactate dehydrogenase are shown. C~
Statistics Data are expressed as mean(SEM) and n refers to the number of experiments. Except for data presented in table 11, where a paired t test was used, statistical analysis at p
