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Prostacyclin-inhibition of lysine accumulation by the rat left ventricle
Comp.Biochem.Physiol.Vol. 89A, No. 3, pp. 305-308, 1988 Printed in Great Britain
0300-9629/88$03.00+ 0.00 Pergamon Press plc
PROSTACYCLIN-INHIBITION OF LYSINE ACCUMULATION BY THE RAT LEFT VENTRICLE MICHEL G.
NASSER,*JUMANAMARRUSH,CAMILLEF. NASSAR,NICOLAS W.
SHAMMAS,
ABM) JURJUS and M. ZGUHAIR HABBAL
Departments of Physiology, Medicine, Laboratory Medicine and Human Morphology, Faculty of Medicine and American University Medical Center, American University of Beirut, Beirut, Lebanon (Received 21 January 1987) Abstract-l. Left ventricular slices of male SpragueDawley rats were incubated with a fixed concentration of 0.5 rCi/ml 3H-lysine and several concentrations of unlabelled lysine ranging from 0.2 to 5.0 mM in control and prostacylin-treated experiments. The time of incubation ranged from 0.5 to 90 min. 2. Left ventricular slices, were cut to have an optimal thickness of 0.47 + 0.09 mm. 3. Lysine was taken up against a concentration gradient. Saturation was reached at 0.5 mM and steady state accumulation of lysine was attained within 60min. 4. Prostacyclin in concentrations ranging from 1.2 x IO-* to 4.8 x IO-* M inhibited lysine transport in left ventricular slices significantly (P < 0.01).
INTRODUCTION Uptake and utilization of amino acids by the heart have been widely investigated but the mechanisms that control their movement across cell membranes are not fully understood. It is well documented that myocardial cells take up amino acids against a concentration gradient thus establishing an intracellular concentration several times larger than extracellular (Morgan et al., 1971; ScharfT and Wool, 1965). Arterial concentration of free amino acids does affect this concentration and any rise even within the physiologic range is associated with a disproportionate increase in their extraction (Bing et al., 1954). Thus, the carrier-mediated transport is active enough to secure at all times an adequate supply of amino acids to cardiac muscle cells: a fact which enables the heart to maintain a normal structure and assume an optimal function. Among essential amino acids, lysine is one that has a specially high rate of uptake by the heart. In particular, lysine is concentrated by cardiac myosin twice as much as any other essential amino acid (Morgan et al., 1971). In addition it participates in cardiac growth (Banos et al., 1970) and functions as a cation in potassium deficiency to help maintain myocardial ion balance (Bckel et al., 1954). Endogenous hormones, such as insulin, growth hormone and prostaglandins, modulate amino acid transport at sacrolemmal membranes (Hjalmarson et al., 1969; Mitrovio et al., 1981; Wildenthal et al., 1976; Rodemann and Goldberg, 1982). Incorporation of ‘H-glycine into bovine articular chondrocytes was inhibited by most protanoids; however, prostacyclin was shown unexpectedly to enhance uptake of ‘H-glycine into the same cells (Mitrovio et al., 1981). It was therefore tempting to investigate whether prostacyclin plays a role in the transport of other amino acids across myocardial cells, in particu*To whom correspondence
should be addressed.
lar lysine because of its important taining cell structure integrity.
function in main-
MATERIALSANDMETHODS Experimental animals and tissue preparation Male Sprague-Dawley rats, weighing from 15@-250g were anesthetized with intraneritoneal sodium oentobarbital at a dosage of 6 mg/lOO g body wt. The ches; was opened, and the left ventricle ranidlv excised and placed in Krebs buffer cooled to 0-4”C. _ _ The left ventricle was washed of blood, trimmed of fat and introduced into a slicer precooled to &4C. A sharp blade (Scientific Apparatus) also precooled to &4C was used for slicing the left ventricle according to the method of Warburg et 01. (Umpreit ef al., 1964). Slice thickness WS calculated and found to be approx. 0.47 f 0.09 mm, a value which is well within the optimal range for diffusion and transport of oxygen and substrates (Umbreit et al., 1964; Pearson et al., 1949). All slices were incubated in Krebs-Hensleit buffer at 37°C under continuous bubbling of a gas mixture of 95% 0, and 5% COr, thereby achieving a PO, of 750 mmHg (Utley and Ashleigh, 1982). The pH of the solution was adjusted to 7.4 with 3 N HCl and it was noted to remain unchanged during bubbling with 0, and COz. Cell viability and membrane integrity were tested and confirmed by measuring tissue oxygen consumption and performing microscopic studies respectively. A YSI Model 53 biological oxygen monitor using a specially designed Clark type polarographic electrode was used to measure tissue oxygen consumption. Oxygen consumption by left ventricular slices was found comparable to data published by Pearson et al. (1949) suggesting that we are dealing indeed with alive cells, capable of performing physiologically. Incubation studies “C-Inulin, cold and/or labeled ‘H-lysine and prostacyclin were added to the incubation medium according to a preset protocol. At the end of all experiments, slices were removed from the incubation medium, blotted with No. 1 Whatman filter papers, weighed on a Mettler balance and extracted in water for 12 hr. Samples of the extracts, representing total
MKHEL G. NAZZRR er al.
306
intracellular and extracellular cpm, and incubation media were counted for their 3H and r4c content in a Rack Beta liquid Scintillation Counter, LKB-Wallac (Model 43), using Bray’s solution as a scintillation liquid, Tbe tissue water content was calculated from the difference between the wet and dry wt of slices ahowed to dry for 24 hr at 98°C (Hajjar et al., 1973). The extracellular space was determined using “C-imtlin by a method similar to that described by Hajjar and Curran (1970).
P
0
Intracellular uptake of lysine was expressed as the i4C-lysine distribution ratio [intraceihrlar counts per minute (cpm) per &counts per minute per ~1 in the incubation medium]. This ratio indicates the extent to which lysine is extracted from the extracellular space. It is assumed that the concentration of ‘*C-1ysine in the extracelhnar water at the end of the incubation period was the same as that of the in~bation medium. B&x was calculated from tissue uptake of )H-&sine aftrt correction of the inulin space as described by Hajjar and Curran (1970). The mean rate of lysine uptake by left ventricular slices was calculated as intracellular cpm/min. Time-related iysine accumtdation by rat left ventricular slices 3H-Lysine (Amersham Radio Chemical Center) was incubated with heart slices in Krebs-Hensleit bt&er at a concentration of O.OSrCi/ml. The time of incubation ranged between 30 set and 9Omin. Incubation experiments and intracellular determination of ‘*C-lysine were performed as described above. Efeci of ex~racel~~at iysine ro~c~~raiio~ on &sine accumulation. The effect of extracellular lysine concentration on the distribution ratio of lysine was investigated using a range of concentrations of unlabelled lysine (0.2-S.OmM) to which was added a constant concentration of 0.05 @i/ml of labelled lysine. Ptepararationof the soWon of ~ros~acyclin The solution of prostacyclin was prepared as described by Lippton et a/.(1979). A 2.67 x lo-‘M stock solution was made by adding 1.OOmg of prostacyclin (Sigma Chemical Co.), used as the sodium salt, to 100 ml of a 20 mM Tris base buffer previously adjusted to pH 9.00. The solution was kept frozen in a dark bottle under nitrogen gas. All steps involved in the preparation of prostacyclin solution were processed in crushed ice. The solution as such was stable for
several weeks.
i
1
J
IO
20
Time
L
I 60
1 M of
incubation
so fmtnl
Fig. 1. Effect of time on accumulation of lysine in rat left ventricle. Lysine aaxnnulation approaches a steady state at 60 min of incubation. At this point the in/out ratio is 1.1. Each point represents the mean of at least 10 experiments. Bars represent f SEM. Time-related lysine accumulation in rat left ventricular slices In studying the accumulation of lysine as a function of time, the duration of incubation ranged from 0.5 to 90min. The results of these experiments are presented in Fig. 1. Lysine a~umula~on approaches steady state at 64 min of incubation. At this point the in/out ratio is 1: 1. Accordingly all experiments were performed at 60min. When lysine uptake is expressed as the mean rate of uptake per minute, it is shown to fall steadily as it approaches steady state (Fig. 2). E$ects of ex~racel~ular iysine c~~ce~~rati~~ on
lysine accumulation The effects of extracellular lysine cconcentration on intracellular lysine accumulation are shown in Fig. 3.
SO
2
60
E
&
70
z*
60
,P 4 e
Efict of PGI, on iysine accumulation Left ventricular slices were incubated with increasing conczmtrations of PG& (1.2 x 10-s M-4.7 x 10-s M) in the presence of a constant concentration of ?H-Iysine (0.05 ~~i/rnl) and unlabelled lysine (0.5 mM). Statistical analysis The statistical significance of the difference between means was tested using Student’s f-test.
‘S 45 6 r
50
40
5
30
f
20
RESULTS Determination
of rhe extracellular
space (ECS)
ECS was found to be 53.5% of the wet wt, a value close to that found by Morgan et al. (1961) using sorbitol.
Time
of
incubation
(min)
Fig. 2. Effect of time on mean rate of lysine accumulation. A steady fall in the rate of uptake of lysine is noted as myocardial cells approach steady state. Each point represents the mean of at least 10 experiments. Bars represent f SEM.
Lysine accumulation
4.,io 0 0.2 0.5
I .o
Extracellutar
2.0
3.0
lyslns concentration
(mM)
Fig. 3. F&et of extracellular lysine concentration on lysine accumulation in rat left ventricle. Each point represents the mean of at least 10 experiments. Bars represent f SEM.
The ability of myocardial cells to accumulate lysine is evident at all concentrations. At the top value of 5.0 mM extracellular lysine concentration, the distribution ratio is nearly 2.0 and this is statistically significant from the distribution ratio of 1.5 obtained at 0.5 mM (P < 0.01). Effect of prostacyclin on lysine accumulation The effects of increasing extracellular concentration of prostacyclin on lysine accumulation from a starting PCS, con~n~a~on of 1.2 x lo-’ to a final concentration of 4.7 x 10e8 M are shown in Fig. 4. There was consistent inhibition of lysine accumulation by prostacyclin. The calculated per cent inhibition of lysine accumulation was statistically ~g~fi~nt from control at the initial con~ntration of prostacyclin (P c 0.01). This initiai inhibition of lysine accumulation by prostacyclin was not affected significantly by increasing prostacyclin concentration suggesting an all-or-none effect of prostacyclin. DISCUSSION Left ventricular slices were cut to assume an optimal thickness of 0.47 + 0.09 mm according to the tissue slicing technique of Warburg et af. (Umbreit et al., 1964). At this thickness no damage to the cell membrane occurs and transport and/or diffusion of
5 051, { , , ] 0
1.0
20
3.0
4.0
5.0
Concentration of prostacyclin in the incubation medium (x10-‘M)
Fig. 4. Effect of prostacyclin on intracellular accumulation ofiysine in rat lefi ventride. A consistent inhibition of lysine accumulation by PGI, is noted. This inhibitory effect is an all or none effeci. Each point represents the mek of at least 10 experiments. Bars represent f SEM.
by left ventricle slices
301
substrates and oxygen can take place readily (Umb&t ef al., 1964; Pearson et al., 1949). The integrity of membrane structure was assessed at microscopy and was shown to be well preserved. Cell viability and function were documented by measuring oxygen consumption in a set of experiments. Left ventricular slices were found to consume oxygen within the normal range (Pearson et al., 1949) indicating that they were thriving on oxidative metabolism. In our experiments, the incubation medium approached as physiobgic an environment as possible. It included all substrates and ions that normally surround myocardial cells. Repeated measurements of osmolarity in aliquots of the medium were 290mosm consistently. On the other hand, maintenance of constant and physiologic pH and temperature as well as an optimal POz of 750 mmHg and oxygen content of 140~1 in the incubation medium throughout the experimental period contributed largely to the stability of the preparation (Utley and Ashleigh, 1982). Under the above nearly ideal environmental conditions, tissue metabolic measurements can reasonably be labelled as qualitatively valid if not quantitatively. At any rate, measurements of changes are safe and reliable. Extracellular space (KS) was measured using “C-inulin, a saccharide molecule which is readily and fully miscible in and exclusively confined to the ECS (Rosenberg et al., 1%2a,b). In our experiments, measurements of the ECS were comparable in magnitude to those obtained by others whether inulin or sorbitol was used (Morgan et al., 1961) Lysine a~umulation by left ventricular slices should be interpreted in the light of the following assumptions: 1. It is assumed that lysine remains free in solution or is at least in an exchangeable form throu~out the experiment. This assumption overlooks alternate fates of the molecule of lysine. These fates include utilization of lysine in oxidative metabolism as an energy provider and incorporation and recycling into proteins or other nitrogen compounds. All these alternate pathways of lysine tend to decrease its concentration in the cytoplasm and result in higher in/out distribution ratio. On the other hand, release of lysine into cytoplasm following degradation of proteins or other nitrogenous compounds will result in a decrease in the in/out distribution ratio through inhibition of uptake. Clearly the balance of these two forces can be a source of error: (a) if they do take place to a significant extent, (b) if they do not cancel each other. Experiments done on turtle ventricles (Hajjar et al., 1973) showed lysine to be 90% free. Although this finding could be species dependent, it is considered as indirect evidence that the 3H-label that we are counting represents indeed actual free lysine. 2. It is also assumed that changes in labelled lysine reflect changes in both labelled and unlabelled lysine. This is a safe assumption because hot and cold lysine are readily miscible as their chemical characteristics are identical. In our experiments lysine was taken up by the heart against a concentration gradient judging by the
MICHELG. NAXZR er al.
308
in/out distribution ratio which was consistently above 1.0, approaching 2.0 at the higher extracellular concentration of 50mM. Our results are similar to other experiments done in isolated cat and intact rat diaph~~ (Manchester and Young, 19601, brain tissue slices (Neame, 1961), rat kidney cortex slices (Rosenberg et al., 1962a,b), rabbit lens (Cotlier, 1971), turtle ventricle (Hajjar et al., 1973) and in studies on intestinal absorption and renal tubular reabsorption (Bellas, 1960). However concentration gradients against which lysine is taken up by mycocardial cells are lower in our experiments than those reported by others using perfused hearts (Manchester, 1963). Our data indicate that we are dealing with a carrier-mediated process with possibly an active step characterized by a saturation phenomenon. Several workers (Bing er al., 1954; Banos et al., 1978; Banos et al., 1970) have shown that amino acids enter the myocardium by a carrier-mediated process of limited capacity rather than by diffusion. The effect of prostacyclin on lysine accumulation by heart slices was evident in all experiments. There was consistent inhibition of lysine transport but the in/out distribution ratio did not decrease below 1.0. Interestingly the effects of prostacyclin appear to outlive its half-life. At pH 7.4 or lower and at normal body temperature, the half-life of prostacyclin is about 3 min following which it degrades into 6 ketoPGF,a, a biologically stable but inactive product (Stehie, 1982). This inhibition of lysine a~um~ation by left ventricular slices might be due either to in~bition of the active part of lysine transport such as ATPase and/or adenylate cyclase systems, or to a competitive inhibitory effect of PGI, best expressed by irreversible changes in the conformation of lysine receptors. A third possibility could be that prostacyclin binds to receptors of its own at the cell membrane making lysine carriers unreceptive. In conclusion, prostacyclin exerts an inhibitory effect on lysine transport in left ventricular slices. Further investigations are needed to clarify the mechanism of this effect.
as a muscle cation in potassium deficiency. Arch. Biochetn. Biophys. 52, 293-294.
Haiiar J. J. and Curran F. P. (1970) Characteristics of the a&no acid transport system in the mucosal border of rabbit ileum. J. gen. Physioi. 56, 673-691. Hajjar J. J., Khuri R. N. and Bizri H. (1973) Lysine transport in turtle ventricle-I. Effect of ouabain and extracellular ions on lysine accumulation. Comp. Eiothem. Physiol. 46A, 45-46. Hjalmarson A. er al. (1969) Effects of growth hormone and insulin on amino acid transport in perfused rat heart. Am. J. Physioi. 217, 1795-1802.
Lippton H. L., Kadowitz, P. J., Pa&an P. W., Million B. T., Nelson P. K., Feigen L. P., Chapnick B. M. and Hyman A. L. (1979) Cardiovascular actions of PGI, in the cat. Arch. Int. Pharmacol. 241, 121-130. Manchester K. L. (1963) Insulin and incorporation of amino acids into protein of perfused rat heart-2. Accumulation and incorporation studies with the perfused rat heart. Biochem. J. 89, 202. Manchester K. L. and Young F. G, (1960) The effect of insulin in vitro on the accumulation of amino acids by isolated rat diaphragm. Biochem. J. 75, 487495. Mitrovio D. et al. (1981) Effects of various prostanoids on the in oitro metabclism of bovine articular chondrocytes. Prostaglandins 22, 4995 11.
Morgan H., Earl D., Broadus A., Wolpert E. I%., Giger K. E. and Jefferson L. S. (1971) Regulation of protein synthesis in heart muscle. I. Effect of amino acid levels on protein synthesis. J. biol. Chem. 246, 2152-2162. Mornan H. E.. Henderson M. J.. Reaen D. M. and Park C.-R. (1961) Regulation of g&o~ uptake in muscle. 1. The effects of insulin and anoxia on glucose transport and ohosphorvlation in the isolated, perfused heart of normal rats. i biol. Chem. 236,253-261. Neame K. D. (1961) Uptake of amino acids by mouse brain slices. J. Neurochem. 6, 358-366. Pearson 0. H., Hastings A. B. and Bunting H. (1949) Metabolism of cardiac muscle. Utilization of “C-1abelled nvruvate and acetate bv rat heart slices. Am. J. Phvsiof. ij, 251-260. Rodemann H. P. and Goldberg A. L. (1982) Arachidonic acid, prostaglandin & and Fza,influence rats of protein turnover in skeletal and cardiac muscle. J. biol. Chem. 257, 1632-1638.
Rosenberg L. E., Downing S. J. and Segal S. (1962a) Competitive inhibition of diabasic amino acid transport in rat kidney. J. biol. Chem. 237, 2265-2270. Rosenberg L. E., Downing S. J. and Segal S. (1962b) Extracellular space estimation in rat kidney slices using ‘C saccharides and phlorixin. Am. J. Physiol. ZOZ, 800-804.
REFERENCES
Banos G., Daniel P. M., Moorhouse S. R. and Pratt D. E. (1970) The entry of amino acids into the myocardium of the growing rat in uivo. J. Physiol. 210, 149.
Banos G., Daniel P. M,, Moorhouse S. R., Pratt D. E. and Wilson P. A. (1978) The inllux of amino acids into the heart of the rat. J. Physiol. 280, 471-486. Bellas D. (1960) Absorption of r&sine in the small intestine of rats. Experientia 16, 367-368. Bing R. J., Siegel A., Ungar I. and Gilbert M. (1954) Metabolism of the human heart: studies on fat, ketone and amino acid metabolism. Am. J. Med. 16, 504-515. Cotliar E. (1971) Lysine transport and protein incorporation by the lens. 3~~~~. biophys A&z 241, 798-806. Eckel R. E., Pope C. E. and Norris J. E, C. (1954) Lysine
Scharff R. and Wool i. G. (1965) Accumutation of ammo acid into muscle of perfused rat heart: Effect of insulin. Biochem. J. 97, 257-271.
Stehle R. G. (1982) Physical chemistry, stability and handling of prostaglandins I$, Fs, and I,: A critical summary. Meth. Enzymol. 86, 436458.
Umbreit W., Burris R. and Stauffer 3. (1964) Delhi Preparing Animal T&sues in ~~o~~r~c
Tec~jq~s,
of
4th
edn, pp. 114-117. Burgess, Minneapolis. Utley J. R. and Ashleigh E. A. (1982) Fluorocarbons and synthetic blood: Capabilities and future uses. In Pathophysiology and Techniques of Cardiopulmonary Bypass,
Vol. 1, p. 14. Waverly Press, UK. Wildenthal K., Grit&t E. E. and lngwall J. S. (1976) Hormonal control of cardiac protein and amino acid balance. Circ. Res. 38, 138-142.
0300-9629/88$03.00+ 0.00 Pergamon Press plc
PROSTACYCLIN-INHIBITION OF LYSINE ACCUMULATION BY THE RAT LEFT VENTRICLE MICHEL G.
NASSER,*JUMANAMARRUSH,CAMILLEF. NASSAR,NICOLAS W.
SHAMMAS,
ABM) JURJUS and M. ZGUHAIR HABBAL
Departments of Physiology, Medicine, Laboratory Medicine and Human Morphology, Faculty of Medicine and American University Medical Center, American University of Beirut, Beirut, Lebanon (Received 21 January 1987) Abstract-l. Left ventricular slices of male SpragueDawley rats were incubated with a fixed concentration of 0.5 rCi/ml 3H-lysine and several concentrations of unlabelled lysine ranging from 0.2 to 5.0 mM in control and prostacylin-treated experiments. The time of incubation ranged from 0.5 to 90 min. 2. Left ventricular slices, were cut to have an optimal thickness of 0.47 + 0.09 mm. 3. Lysine was taken up against a concentration gradient. Saturation was reached at 0.5 mM and steady state accumulation of lysine was attained within 60min. 4. Prostacyclin in concentrations ranging from 1.2 x IO-* to 4.8 x IO-* M inhibited lysine transport in left ventricular slices significantly (P < 0.01).
INTRODUCTION Uptake and utilization of amino acids by the heart have been widely investigated but the mechanisms that control their movement across cell membranes are not fully understood. It is well documented that myocardial cells take up amino acids against a concentration gradient thus establishing an intracellular concentration several times larger than extracellular (Morgan et al., 1971; ScharfT and Wool, 1965). Arterial concentration of free amino acids does affect this concentration and any rise even within the physiologic range is associated with a disproportionate increase in their extraction (Bing et al., 1954). Thus, the carrier-mediated transport is active enough to secure at all times an adequate supply of amino acids to cardiac muscle cells: a fact which enables the heart to maintain a normal structure and assume an optimal function. Among essential amino acids, lysine is one that has a specially high rate of uptake by the heart. In particular, lysine is concentrated by cardiac myosin twice as much as any other essential amino acid (Morgan et al., 1971). In addition it participates in cardiac growth (Banos et al., 1970) and functions as a cation in potassium deficiency to help maintain myocardial ion balance (Bckel et al., 1954). Endogenous hormones, such as insulin, growth hormone and prostaglandins, modulate amino acid transport at sacrolemmal membranes (Hjalmarson et al., 1969; Mitrovio et al., 1981; Wildenthal et al., 1976; Rodemann and Goldberg, 1982). Incorporation of ‘H-glycine into bovine articular chondrocytes was inhibited by most protanoids; however, prostacyclin was shown unexpectedly to enhance uptake of ‘H-glycine into the same cells (Mitrovio et al., 1981). It was therefore tempting to investigate whether prostacyclin plays a role in the transport of other amino acids across myocardial cells, in particu*To whom correspondence
should be addressed.
lar lysine because of its important taining cell structure integrity.
function in main-
MATERIALSANDMETHODS Experimental animals and tissue preparation Male Sprague-Dawley rats, weighing from 15@-250g were anesthetized with intraneritoneal sodium oentobarbital at a dosage of 6 mg/lOO g body wt. The ches; was opened, and the left ventricle ranidlv excised and placed in Krebs buffer cooled to 0-4”C. _ _ The left ventricle was washed of blood, trimmed of fat and introduced into a slicer precooled to &4C. A sharp blade (Scientific Apparatus) also precooled to &4C was used for slicing the left ventricle according to the method of Warburg et 01. (Umpreit ef al., 1964). Slice thickness WS calculated and found to be approx. 0.47 f 0.09 mm, a value which is well within the optimal range for diffusion and transport of oxygen and substrates (Umbreit et al., 1964; Pearson et al., 1949). All slices were incubated in Krebs-Hensleit buffer at 37°C under continuous bubbling of a gas mixture of 95% 0, and 5% COr, thereby achieving a PO, of 750 mmHg (Utley and Ashleigh, 1982). The pH of the solution was adjusted to 7.4 with 3 N HCl and it was noted to remain unchanged during bubbling with 0, and COz. Cell viability and membrane integrity were tested and confirmed by measuring tissue oxygen consumption and performing microscopic studies respectively. A YSI Model 53 biological oxygen monitor using a specially designed Clark type polarographic electrode was used to measure tissue oxygen consumption. Oxygen consumption by left ventricular slices was found comparable to data published by Pearson et al. (1949) suggesting that we are dealing indeed with alive cells, capable of performing physiologically. Incubation studies “C-Inulin, cold and/or labeled ‘H-lysine and prostacyclin were added to the incubation medium according to a preset protocol. At the end of all experiments, slices were removed from the incubation medium, blotted with No. 1 Whatman filter papers, weighed on a Mettler balance and extracted in water for 12 hr. Samples of the extracts, representing total
MKHEL G. NAZZRR er al.
306
intracellular and extracellular cpm, and incubation media were counted for their 3H and r4c content in a Rack Beta liquid Scintillation Counter, LKB-Wallac (Model 43), using Bray’s solution as a scintillation liquid, Tbe tissue water content was calculated from the difference between the wet and dry wt of slices ahowed to dry for 24 hr at 98°C (Hajjar et al., 1973). The extracellular space was determined using “C-imtlin by a method similar to that described by Hajjar and Curran (1970).
P
0
Intracellular uptake of lysine was expressed as the i4C-lysine distribution ratio [intraceihrlar counts per minute (cpm) per &counts per minute per ~1 in the incubation medium]. This ratio indicates the extent to which lysine is extracted from the extracellular space. It is assumed that the concentration of ‘*C-1ysine in the extracelhnar water at the end of the incubation period was the same as that of the in~bation medium. B&x was calculated from tissue uptake of )H-&sine aftrt correction of the inulin space as described by Hajjar and Curran (1970). The mean rate of lysine uptake by left ventricular slices was calculated as intracellular cpm/min. Time-related iysine accumtdation by rat left ventricular slices 3H-Lysine (Amersham Radio Chemical Center) was incubated with heart slices in Krebs-Hensleit bt&er at a concentration of O.OSrCi/ml. The time of incubation ranged between 30 set and 9Omin. Incubation experiments and intracellular determination of ‘*C-lysine were performed as described above. Efeci of ex~racel~~at iysine ro~c~~raiio~ on &sine accumulation. The effect of extracellular lysine concentration on the distribution ratio of lysine was investigated using a range of concentrations of unlabelled lysine (0.2-S.OmM) to which was added a constant concentration of 0.05 @i/ml of labelled lysine. Ptepararationof the soWon of ~ros~acyclin The solution of prostacyclin was prepared as described by Lippton et a/.(1979). A 2.67 x lo-‘M stock solution was made by adding 1.OOmg of prostacyclin (Sigma Chemical Co.), used as the sodium salt, to 100 ml of a 20 mM Tris base buffer previously adjusted to pH 9.00. The solution was kept frozen in a dark bottle under nitrogen gas. All steps involved in the preparation of prostacyclin solution were processed in crushed ice. The solution as such was stable for
several weeks.
i
1
J
IO
20
Time
L
I 60
1 M of
incubation
so fmtnl
Fig. 1. Effect of time on accumulation of lysine in rat left ventricle. Lysine aaxnnulation approaches a steady state at 60 min of incubation. At this point the in/out ratio is 1.1. Each point represents the mean of at least 10 experiments. Bars represent f SEM. Time-related lysine accumulation in rat left ventricular slices In studying the accumulation of lysine as a function of time, the duration of incubation ranged from 0.5 to 90min. The results of these experiments are presented in Fig. 1. Lysine a~umula~on approaches steady state at 64 min of incubation. At this point the in/out ratio is 1: 1. Accordingly all experiments were performed at 60min. When lysine uptake is expressed as the mean rate of uptake per minute, it is shown to fall steadily as it approaches steady state (Fig. 2). E$ects of ex~racel~ular iysine c~~ce~~rati~~ on
lysine accumulation The effects of extracellular lysine cconcentration on intracellular lysine accumulation are shown in Fig. 3.
SO
2
60
E
&
70
z*
60
,P 4 e
Efict of PGI, on iysine accumulation Left ventricular slices were incubated with increasing conczmtrations of PG& (1.2 x 10-s M-4.7 x 10-s M) in the presence of a constant concentration of ?H-Iysine (0.05 ~~i/rnl) and unlabelled lysine (0.5 mM). Statistical analysis The statistical significance of the difference between means was tested using Student’s f-test.
‘S 45 6 r
50
40
5
30
f
20
RESULTS Determination
of rhe extracellular
space (ECS)
ECS was found to be 53.5% of the wet wt, a value close to that found by Morgan et al. (1961) using sorbitol.
Time
of
incubation
(min)
Fig. 2. Effect of time on mean rate of lysine accumulation. A steady fall in the rate of uptake of lysine is noted as myocardial cells approach steady state. Each point represents the mean of at least 10 experiments. Bars represent f SEM.
Lysine accumulation
4.,io 0 0.2 0.5
I .o
Extracellutar
2.0
3.0
lyslns concentration
(mM)
Fig. 3. F&et of extracellular lysine concentration on lysine accumulation in rat left ventricle. Each point represents the mean of at least 10 experiments. Bars represent f SEM.
The ability of myocardial cells to accumulate lysine is evident at all concentrations. At the top value of 5.0 mM extracellular lysine concentration, the distribution ratio is nearly 2.0 and this is statistically significant from the distribution ratio of 1.5 obtained at 0.5 mM (P < 0.01). Effect of prostacyclin on lysine accumulation The effects of increasing extracellular concentration of prostacyclin on lysine accumulation from a starting PCS, con~n~a~on of 1.2 x lo-’ to a final concentration of 4.7 x 10e8 M are shown in Fig. 4. There was consistent inhibition of lysine accumulation by prostacyclin. The calculated per cent inhibition of lysine accumulation was statistically ~g~fi~nt from control at the initial con~ntration of prostacyclin (P c 0.01). This initiai inhibition of lysine accumulation by prostacyclin was not affected significantly by increasing prostacyclin concentration suggesting an all-or-none effect of prostacyclin. DISCUSSION Left ventricular slices were cut to assume an optimal thickness of 0.47 + 0.09 mm according to the tissue slicing technique of Warburg et af. (Umbreit et al., 1964). At this thickness no damage to the cell membrane occurs and transport and/or diffusion of
5 051, { , , ] 0
1.0
20
3.0
4.0
5.0
Concentration of prostacyclin in the incubation medium (x10-‘M)
Fig. 4. Effect of prostacyclin on intracellular accumulation ofiysine in rat lefi ventride. A consistent inhibition of lysine accumulation by PGI, is noted. This inhibitory effect is an all or none effeci. Each point represents the mek of at least 10 experiments. Bars represent f SEM.
by left ventricle slices
301
substrates and oxygen can take place readily (Umb&t ef al., 1964; Pearson et al., 1949). The integrity of membrane structure was assessed at microscopy and was shown to be well preserved. Cell viability and function were documented by measuring oxygen consumption in a set of experiments. Left ventricular slices were found to consume oxygen within the normal range (Pearson et al., 1949) indicating that they were thriving on oxidative metabolism. In our experiments, the incubation medium approached as physiobgic an environment as possible. It included all substrates and ions that normally surround myocardial cells. Repeated measurements of osmolarity in aliquots of the medium were 290mosm consistently. On the other hand, maintenance of constant and physiologic pH and temperature as well as an optimal POz of 750 mmHg and oxygen content of 140~1 in the incubation medium throughout the experimental period contributed largely to the stability of the preparation (Utley and Ashleigh, 1982). Under the above nearly ideal environmental conditions, tissue metabolic measurements can reasonably be labelled as qualitatively valid if not quantitatively. At any rate, measurements of changes are safe and reliable. Extracellular space (KS) was measured using “C-inulin, a saccharide molecule which is readily and fully miscible in and exclusively confined to the ECS (Rosenberg et al., 1%2a,b). In our experiments, measurements of the ECS were comparable in magnitude to those obtained by others whether inulin or sorbitol was used (Morgan et al., 1961) Lysine a~umulation by left ventricular slices should be interpreted in the light of the following assumptions: 1. It is assumed that lysine remains free in solution or is at least in an exchangeable form throu~out the experiment. This assumption overlooks alternate fates of the molecule of lysine. These fates include utilization of lysine in oxidative metabolism as an energy provider and incorporation and recycling into proteins or other nitrogen compounds. All these alternate pathways of lysine tend to decrease its concentration in the cytoplasm and result in higher in/out distribution ratio. On the other hand, release of lysine into cytoplasm following degradation of proteins or other nitrogenous compounds will result in a decrease in the in/out distribution ratio through inhibition of uptake. Clearly the balance of these two forces can be a source of error: (a) if they do take place to a significant extent, (b) if they do not cancel each other. Experiments done on turtle ventricles (Hajjar et al., 1973) showed lysine to be 90% free. Although this finding could be species dependent, it is considered as indirect evidence that the 3H-label that we are counting represents indeed actual free lysine. 2. It is also assumed that changes in labelled lysine reflect changes in both labelled and unlabelled lysine. This is a safe assumption because hot and cold lysine are readily miscible as their chemical characteristics are identical. In our experiments lysine was taken up by the heart against a concentration gradient judging by the
MICHELG. NAXZR er al.
308
in/out distribution ratio which was consistently above 1.0, approaching 2.0 at the higher extracellular concentration of 50mM. Our results are similar to other experiments done in isolated cat and intact rat diaph~~ (Manchester and Young, 19601, brain tissue slices (Neame, 1961), rat kidney cortex slices (Rosenberg et al., 1962a,b), rabbit lens (Cotlier, 1971), turtle ventricle (Hajjar et al., 1973) and in studies on intestinal absorption and renal tubular reabsorption (Bellas, 1960). However concentration gradients against which lysine is taken up by mycocardial cells are lower in our experiments than those reported by others using perfused hearts (Manchester, 1963). Our data indicate that we are dealing with a carrier-mediated process with possibly an active step characterized by a saturation phenomenon. Several workers (Bing er al., 1954; Banos et al., 1978; Banos et al., 1970) have shown that amino acids enter the myocardium by a carrier-mediated process of limited capacity rather than by diffusion. The effect of prostacyclin on lysine accumulation by heart slices was evident in all experiments. There was consistent inhibition of lysine transport but the in/out distribution ratio did not decrease below 1.0. Interestingly the effects of prostacyclin appear to outlive its half-life. At pH 7.4 or lower and at normal body temperature, the half-life of prostacyclin is about 3 min following which it degrades into 6 ketoPGF,a, a biologically stable but inactive product (Stehie, 1982). This inhibition of lysine a~um~ation by left ventricular slices might be due either to in~bition of the active part of lysine transport such as ATPase and/or adenylate cyclase systems, or to a competitive inhibitory effect of PGI, best expressed by irreversible changes in the conformation of lysine receptors. A third possibility could be that prostacyclin binds to receptors of its own at the cell membrane making lysine carriers unreceptive. In conclusion, prostacyclin exerts an inhibitory effect on lysine transport in left ventricular slices. Further investigations are needed to clarify the mechanism of this effect.
as a muscle cation in potassium deficiency. Arch. Biochetn. Biophys. 52, 293-294.
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Lippton H. L., Kadowitz, P. J., Pa&an P. W., Million B. T., Nelson P. K., Feigen L. P., Chapnick B. M. and Hyman A. L. (1979) Cardiovascular actions of PGI, in the cat. Arch. Int. Pharmacol. 241, 121-130. Manchester K. L. (1963) Insulin and incorporation of amino acids into protein of perfused rat heart-2. Accumulation and incorporation studies with the perfused rat heart. Biochem. J. 89, 202. Manchester K. L. and Young F. G, (1960) The effect of insulin in vitro on the accumulation of amino acids by isolated rat diaphragm. Biochem. J. 75, 487495. Mitrovio D. et al. (1981) Effects of various prostanoids on the in oitro metabclism of bovine articular chondrocytes. Prostaglandins 22, 4995 11.
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