The use of Langendorff perfused guinea-pig heart to study the efflux of amino acids from heart cells.

Abstract

The inclusion of the 0-amino acid taurine or any of the nonessential amino acids glutamate, aspartate and glutamine in the perfusate, protects the heart during cardiac insults [l-41. The cardioprotective action of taurine may be related to an effect on intracellular Na' and Ca'+-mobilisation [ 11. me cardioprotective action of exogenous glutamate and aspartate has been explained in terms of increasing intracellular levels of these amino acids. This in turn enhances myocardial ATP production during ischaemia and reperfusion, and in the case of glutamate improves the clearance of lactate by the transamination of pyruvate to alanine [5,6]. The mechanism for glutamine's cardioprotective action is not known although its intracellular anabolic activity may be important [7]. This metabolic interpretation of the action of non-essential amino acids neglects the amino acid transport which has for many years been known to influence tissue metabolism and nutrition [8,9). We have recently shown that in addition to the metabolic utilisation of these amino acids, their transport may also influence the extent of myocardial protection [10,11], possibly by forming a rate limiting step in providing substrates for metabolic activity or by regulating intracellular ionic species (e.g. Na' and Ca). Most studies investigating the cardioprotective action of exogenous amino acids have used isolated animal hearts perfused with media devoid of amino acids, creating a gradient favouring an efflux of amino acids. In one such model, the Langendorff guineapig heart perfused with Tyrode solution, we have shown a time dependent fall in the intracellular free amino acid pool [ 10,121. Furthermore exposure of these hearts to different experimental conditions induced significant changes in amino acids present at high concentration, namely: taurine, glutamine, glutamate, aspartate and alanine [10,12]. This was interpreted as resulting from an efflux although metabolic changes were also implicated; however, the extent of efflux and its contribution to tissue levels were not assessed. In this work we report changes in taurine and principal non-essential amino acids in Langendorff guinea-pig heart perfused with Tyrode solution devoid of amino acids. The total release and rate of appearance of amino acids in the effluent were also determined. Hearts were perfused at a constant flow at 37°C as described previously [12]. At the end of the experiments (after 1, 15, 30, 60 and 90 minutes of perfusion) hearts were removed for ATP and amino acids determination [13]. The procedure for tissue preparation for free amino acid determination is essentially similar to that reported earlier [13]. Because amino acids in the effluent were diluted, only amino acids present at high concentration could be detected using flus technique. To concentrate the sample would have increased the salt concentration (present in Tyrode solution) which would influence the separation and identification of certain amino acids. Subsequently only the amino acids glutamine, alanine, glutamate and taurine were present at sufficient concentration to be detected in the effluent. Perfusion with Tyrode solution provoked a time dependent fall in tissue glutamine, glutamate and taurine. The fall was significant and marked for glutamine. Figure 1A shows that after 90 rnin of perfusion, the hearts' 15.5fl.Opmol.g,' dry weight (n=6) of glutamine has almost disappeared. The fall up to 60 minutes was almost linear at approximately 4pmol.mg~' dry weight.sec-'. The fall in tissue glutamine was accompanied by a corresponding rise in total glutamine release into the effluent. The rate of efflux measured at 5 minutes of perfusion when tissue glutamine is still largely intact (figure 1B) was approximately 2.5pmol.mg-' dry weight.sec-'. Unlike glutamine, tissue taurine fell very slowly, so that after 60 minutes of perfusion the fall was not statistically significant, despite the large concentration gradient present across the sarcolemma [ 11. The small fall in taurine was also accompanied by a release of this amino acid into the effluent. Although alanine was detected in the effluent, there was no change in the intracellular concentration, suggesting that alanine is being synthesised in the cells. Heart cells transaminate glutamate (3.Mo.4) (11.8f1.4) 8 : i : 4 15.3M.7)