- Email: [email protected]
The whole blood-perfused working rat heart in vitro
xvi
30
THE WHOLE BLOOD-PERFUSED WORKING RAT HEART IN VITRO. D.L.Topping, R.P.Trimble. CSIRO. Division of Human Nutrition, Glenthorne Laboratory, O'Halloran Hill, Sth. Australia 5158, Australia. Perfusion is a valuable technique for the study of organ function in vitro under physiological conditions. With heart, two systems are in common use i.e. the retrograde (langendorff) and orthograde (working) preparations. Although the most suitable perfusate would seem to be blood, the perfusate nearly always used in both heart preparations is erythrocyte-free buffer. This is due to the apparent instability of hearts perfused with media containing red blood cells. Studies in this laboratory in rat liver perfused with rat blood have shown that at haematocrits greatly below the physiological, artifacts develope due to anaemic hypoxia. Work has been in progress developing an isolated working rat heart perfused with undiluted rat blood, dialyzed to remove vasoconstrictor factors (Mayes,P.A. & Felts,J.M. (1966). Proc. Eur. Soc. Study Drug Toxicity 7, 16-29). Most of the previously reported problems with blood perfusate would seem to reflect bacterial contamination because when precautions are taken to ensure sterility in preparation of perfusate and apparatus, hearts are obtained which are stable for 90 min as judged by stroke rate and coronary flow. The latter averages 2.4 ml/min/g as compared with 9.6 ml/min/g in hearts perfused with buffer. Aortic blood flow is in the range 35-40 ml/min/g with blood and 20-25 ml/min/g with buffer. The blood perfused working rat heart would appear to be a useful preparation for the study of myocardial metabolism in vitro.
31
RECENT ADVANCES IN CALCIUM DETECTION IN MUSCLE CELLS. I.R. Wendt. Department of Physiology, Monash University, Clayton, 3168, Australia. Whilst the importance of calcium ions in regulating the function of muscle cells can hardly be overstated many aspects of the relationship between muscle function and intracellular Ca ++ concentration, as well as many of the factors responsible for the moment to moment regulation of the sarcoplasmic Ca + + concentration, are not clearly understood. A thorough understanding of these matters will require accurate measurements of the changes that occur in intracellular Ca+ + concentration during normal regulation of cellular function. The past decade has seen considerable advances in the development of techniques for monitoring intracellular Ca ++ concentration in living cells. At the present time there are four main types of indicator which can potentially be used for this purpose. These are Ca + + activated photoproteins, Ca + + selective microelectrodes, metallochromic dyes and fluorescent indicators. Each of these has strengths and weaknesses that differ from those of the others and may determine its applicability for certain purposes. With regard to cardiac muscle the two former approaches have provided most information to date. They have revealed that t~e intracellular Ca ++ concentration in mammalian_~entricular cells is around 2-3 x 10- M at rest and ris~s rapidly to around 1-2 x i0 ~M early in a normal twitch, and to around 4 x 10-~M in a twitch under optimum inotropic conditions. Even this latter concentration is, however, s t i l ~ well below that required for full activation of the myofilaments (around I-2 x 10-~M).
32
CALCIUM ACCUMULATION BY THE SARCOPLASMIC RETICULUM IN MECHANICALLY DISRUPTED CARDIAC MUSCLE. THE EFFECT OF PROCAINE. I.R. Wendt, D.G. Stephenson. Department of Physiology, Monash University, Clayton, 3168 and Department of Zoology, La Trobe University, Bundoora 3083, Australia. Small fragments (diameter 0.05-0.1 mm, length 0.5-1.0 mm) of functionally skinned rat ventricular myocardium were obtained by homogenization in an ATP salt solution containing 50mM EGTA and mounted on a force recording apparatus. The sarcoplasmic reticulum (SR) remains intact and functional in these preparations and accumulates Ca + + when the preparations are placed in suitable Ca++ibuffered loading solutions. ++ The accumulated Ca can subsequently be released by applying 30mM caffeine. This caffeine induced release of Ca ~-~ gives rise the ++ to a transient force response, ~-+ magnitude of which reflects the amount of Ca released. The amount of Ca released ++ d e ~ n d s on both the preceding time spent in the Ca loading solution and the free Ca concentration of that solution. Following an equal time of Ca + + loading, at an equal Ca ++ concentration, the caffeine induced force transient is always substantially larger if procaine (5mM) was p ~ s e n t in the Ca ++ loading solution. This suggests that procaine markedly enhances Ca accumulation by the SR in rat cardiac muscle. The mechanism whereby this occurs is not clear but could involve an inhibition by procaine of a normal ongoing release (or "leak") of Ca + + which may be occurring from the SR.
30
THE WHOLE BLOOD-PERFUSED WORKING RAT HEART IN VITRO. D.L.Topping, R.P.Trimble. CSIRO. Division of Human Nutrition, Glenthorne Laboratory, O'Halloran Hill, Sth. Australia 5158, Australia. Perfusion is a valuable technique for the study of organ function in vitro under physiological conditions. With heart, two systems are in common use i.e. the retrograde (langendorff) and orthograde (working) preparations. Although the most suitable perfusate would seem to be blood, the perfusate nearly always used in both heart preparations is erythrocyte-free buffer. This is due to the apparent instability of hearts perfused with media containing red blood cells. Studies in this laboratory in rat liver perfused with rat blood have shown that at haematocrits greatly below the physiological, artifacts develope due to anaemic hypoxia. Work has been in progress developing an isolated working rat heart perfused with undiluted rat blood, dialyzed to remove vasoconstrictor factors (Mayes,P.A. & Felts,J.M. (1966). Proc. Eur. Soc. Study Drug Toxicity 7, 16-29). Most of the previously reported problems with blood perfusate would seem to reflect bacterial contamination because when precautions are taken to ensure sterility in preparation of perfusate and apparatus, hearts are obtained which are stable for 90 min as judged by stroke rate and coronary flow. The latter averages 2.4 ml/min/g as compared with 9.6 ml/min/g in hearts perfused with buffer. Aortic blood flow is in the range 35-40 ml/min/g with blood and 20-25 ml/min/g with buffer. The blood perfused working rat heart would appear to be a useful preparation for the study of myocardial metabolism in vitro.
31
RECENT ADVANCES IN CALCIUM DETECTION IN MUSCLE CELLS. I.R. Wendt. Department of Physiology, Monash University, Clayton, 3168, Australia. Whilst the importance of calcium ions in regulating the function of muscle cells can hardly be overstated many aspects of the relationship between muscle function and intracellular Ca ++ concentration, as well as many of the factors responsible for the moment to moment regulation of the sarcoplasmic Ca + + concentration, are not clearly understood. A thorough understanding of these matters will require accurate measurements of the changes that occur in intracellular Ca+ + concentration during normal regulation of cellular function. The past decade has seen considerable advances in the development of techniques for monitoring intracellular Ca ++ concentration in living cells. At the present time there are four main types of indicator which can potentially be used for this purpose. These are Ca + + activated photoproteins, Ca + + selective microelectrodes, metallochromic dyes and fluorescent indicators. Each of these has strengths and weaknesses that differ from those of the others and may determine its applicability for certain purposes. With regard to cardiac muscle the two former approaches have provided most information to date. They have revealed that t~e intracellular Ca ++ concentration in mammalian_~entricular cells is around 2-3 x 10- M at rest and ris~s rapidly to around 1-2 x i0 ~M early in a normal twitch, and to around 4 x 10-~M in a twitch under optimum inotropic conditions. Even this latter concentration is, however, s t i l ~ well below that required for full activation of the myofilaments (around I-2 x 10-~M).
32
CALCIUM ACCUMULATION BY THE SARCOPLASMIC RETICULUM IN MECHANICALLY DISRUPTED CARDIAC MUSCLE. THE EFFECT OF PROCAINE. I.R. Wendt, D.G. Stephenson. Department of Physiology, Monash University, Clayton, 3168 and Department of Zoology, La Trobe University, Bundoora 3083, Australia. Small fragments (diameter 0.05-0.1 mm, length 0.5-1.0 mm) of functionally skinned rat ventricular myocardium were obtained by homogenization in an ATP salt solution containing 50mM EGTA and mounted on a force recording apparatus. The sarcoplasmic reticulum (SR) remains intact and functional in these preparations and accumulates Ca + + when the preparations are placed in suitable Ca++ibuffered loading solutions. ++ The accumulated Ca can subsequently be released by applying 30mM caffeine. This caffeine induced release of Ca ~-~ gives rise the ++ to a transient force response, ~-+ magnitude of which reflects the amount of Ca released. The amount of Ca released ++ d e ~ n d s on both the preceding time spent in the Ca loading solution and the free Ca concentration of that solution. Following an equal time of Ca + + loading, at an equal Ca ++ concentration, the caffeine induced force transient is always substantially larger if procaine (5mM) was p ~ s e n t in the Ca ++ loading solution. This suggests that procaine markedly enhances Ca accumulation by the SR in rat cardiac muscle. The mechanism whereby this occurs is not clear but could involve an inhibition by procaine of a normal ongoing release (or "leak") of Ca + + which may be occurring from the SR.