Studies on nuclear amino acid transport and cation content in embryonic myocardium of the chick

Studies on Nuclear Amino Acid Transport and Cation Content in Embryonic Myocardium of the Chick RICHARD L. KLEIN, PhD* CHARLES R. HORTON, BA and ASA THURESON-KLEIN, Fil Lic

Jackson, Mississippi

From the Departmentof Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Miss. This study was supported by research grants from the National Institute of General Medical Sciences (5-RO1-GM15490) and the National Science Foundation (GB-83,18) * Supported by Research Career Program Award 5-KO3-HE05892 from the National Heart Institute. Present address: Department of Physiology, Karolinska Institute, Stockholm, Sweden. Address for reprints: Dr. Richard L. Klein, Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State St., Jackson, Miss. 39216.

300

Nuclei isolated from 12-day embryonic chick heart are capable of transporting a number of amino acids including arginine, alanine and serine. Dicarboxylic amino acids are not transported. Arginine is concentrated in an intranuclear diffusible pool 17 times that expected from a purely passive equilibrium distribution. There is no competition between stereoisomers nor between unlike amino acids, whether or not they are transported. The nuclei bind Ca++ (8 raM) and Na+ (28 mM) in the absence of added cations. M g - - and K+ are not tightly bound. Nuclear Na + content reflects that bound plus that in the extranuclear environment; nuclear K+ can be concentrated in free form above that in the extranuc!ear environment. Nuclear cation content is not appreciably affected by the presence of adenosine triphosphate (ATP) and arginine, except that the M g + + - A T P chelate can not readily permeate the nuclear membrane. Subcellular distribution of Na+ and Ca + - plus Mg++, as revealed by histochemical means, correlates well with direct analytical data. The high nuclear Na+ content can account for only about 50 percent of the total bound Na+ in the myocardium at 12 days of embryonic age. There is evidence of a metabolically dependent transport system for amino acids across the membrane of nuclei isolated from embryonic myocardium of the chick. 1 Specifically, the uptake of alanine appears to be linked to a Mg+*-activated adenosine triphosphate (ATP) phosphohydrolase (International E n z y m e Commission no. E C 3.6.1.3.), which has specific requirements for either Na + or K ÷ and a critical level of Ca ++. Under identical conditions, a number of chemical and physical alterations in the environment have the same characteristic effects on enzymatic hydrolysis of A T P and net uptake of alaninc. The latter can be accumulated into a diffusible pool at 7 to 8 times the concentration present in the suspending medium. Approximately 2 moles of amino acid are transported for each mole of phosphate released from ATP. Alanine uptake was investigated initially on the basis of the earlier report that the accumulation of this amino acid by nuclei isolated from calf thymus required Na ÷ and was probably metabolically dependent. 2 It is now of interest to test the nuclear transport system of the embryonic heart for other amino acids: that is, the basic arginine, because of its guanidine group and high content in nucleohistone: serine, because of its O-C-C-N system and important role as a constituent of active sites on enzymes and pharmacologic receptors; and glutamic and aspartic acids as examples of the dicarboxylic type.

The AmericanJournalof CARDIOLOGY

NUCLEAR AMINO ACIDS AND CATIONS

Because of their relevance to ATP hydrolysis and amino acid transport, analytical and histochemical data on Ca *+. Mg ÷÷, N a ~ and K ÷ contents of nuclei and the myocardium are included.

Methods Nuclei from 12-day embryonic chick hearts were isolated and purified, 3 and the uptake of [14C]labeled amino acids was measured as previously described. 1 A simple experiment was performed to determine how much of the [~4C] L-a arginine content of nuclei existed in a freely diffusible form. Samples of nuclei, from which aliquots were shown to accumulate label, were precipitated with 5 percent trichloroacetic acid. The acid supernatant was alkalinized with ammonia to precipitate any remaining basic protein. The respective precipitates were washed two times with 4 ml of the corresponding acid or alkaline medium before counting. Cation analyses were performed in a Beckman mode] 979 atomic absorption system with a 10-inch recorder. Beckman standards and biologic specimens were dissolved in 0.2 ml concentrated HNO~, 1.0 ml 5 percent Lanthanum-25 percent HC1 and glass-distilled water to 5.0 ml. Readings were taken at 4.227/~(Ca~+), 2.852 A(Mg*+), 5.890 A(Na +) and 7,665 A(K+). The final nuclear purification step was through 2.2 M sucrose containing 3 mM EGTA. Sedimented nuclei were suspended in the various media buffered to p H 7.5 with 20 mM Tris-C1 for 10 minutes at 30C and harvested in polystyrene tubes in a bucket rotor at 1,000 g/5 min at room temperature. The supernatants remained at 30 ° during centrifugation. The total duration of nuclear exposure to suepending media corresponded to that known to result in near maximal accumulation of amino acid. Polystyrene tubes permitted efficient draining of nuclear supernatant and the tube walls were cleaned with damp and then dry gauze to within 5 mm of the nu, clear sediment. Subsequently, the digesting medium was added. Nuclear pellets were visible only as a translucent coating at the bottom of tubes. To correct for adsorbed suspending medium (tube walls and nuclear surface), tubes without nuclei were treated identically and analyzed for extraneous cation; these values were then subtracted from corresponding tubes with nuclei. The correction was significant in reducing the nuclear values only in media with high cation concentration. The histochemical method utilizing K-pyroantimonate to precipitate Na ÷ was a modification of the technique of Komnick 4 (Kaye GI, personal communication). The fixative mixture contained 2 g KSb (OH)6 (K & K Laboratory), 1 g OsO4 (Mercki, 0.1 N K 0 H and acetic acid to pH 7.8 in distilled water to make 100 ml. Experimental tissues we!:e fixed at ice bath temperature for 1 hour, washed with acetate-antimonate mixture without Os04, dehydrated in a series of alcohols and embedded in Epon 812. Grey to

VOLUME 25, MARCH 1970

silver sections were cut on L K B Ultrotomes and viewed in a Zeiss EM9A. To facilitate visualization of antimonate precipitate, sections were not stained for maximal contrast. T h e y were exposed to saturated uranyl acetate for 15 to 30 minutes at room temperature or for 5 to 10 minutes at 60C plus 2 to 5 seconds' exposure to lead citrate. In our experience longer lead staining does not allow unambiguous localization of the finer grained antimonate precipitates. The K-pyro.antimonate experiments were performed on three separate occasions with 4 to 6 specimens in each medium. Some tissues were pre-equilibrated in Ringer's medium ~NaC1 154 raM. KC1 5.4 raM. CaC12 2.2 raM. NaHCO~ 6.0 mM and dextrose 11.1 mMI oxygenated with a mixture of 95 percent 02 and 5 percent CO2 at room temperature (22 t o 24C) to enhance tissue preservation. This temperature permits slow rhythmic contraction and does not affect the Na ~ or K ~ content of the tissue at this age. 5 EGTA. ethyleneglycol bis (~-amin0ethylether)-N, N'-tetraacetie acid. was neutralized with K O H and used at 1.0 and 3.0 raM. Tissue treated with the latter was rinsed with Ca÷*-free Ringer's medium to remove chelated cations and excess E G T A prior to fixation. E G T A could not be employed simultaneously with Os04 without an obvious decrease in blackening of the tissue.

Semiquantitative estimation o] pyroa~timonate precipitate was made by determining the number and size of the particles. Unlabeled mjcrographs were chosen as random from hundreds representing all embeddings. From these, 5 to 10 areas of equal diameter were chosen at random, and the precipitated particles at each of the various subcellular sites were counted and averaged. Four individuals made counts, only 2 of whom were familiar with the experiments and methods. The relative numbers counted were consistent and in agreement among the four. Because of the semiquantitative nature of this estimate, statistical treatment was considered to have little value.

Results Nuclear uptake of arginine: [14C] L-a arginine uptake by the isolated nuclei is entirely analogous qualitatively to that previously reported for alanine3 In the Mg ÷* plus N a ÷ or K + medium, there is an initial rapid uptake reaching a peak at about 10 minutes, followed by a partial loss of isotope by 30 minutes (Fig. 1). The uptake at 10 minutes is 17 times the estimated passive equilibrium value (dashed line). The latter was determined as previously described, i In the basic Mg ÷* medium without added Na t or K*, the average arginine uptake approximates that level expected from a purely passive equilibrium distribution. As with alanine, arginine uptake is influenced by Na* or K ÷ in the medium, with near maximal uptake occurring between 80 to 130 mM and half maximal at 30 to 50 mM of monovalent salt. The optimal concentration of arginine in the external medium is about 3 mM

301

KLEIN ET AL.

for maximal arginine uptake; i.e.. the transport adenosine triphosphatase is saturated. U p t a k e is inhibited by excess Ca ++. removal of Ca *÷ and by ouabain (10 -4 M t. The concentrations of Ca ++ and E G T A (to remove Ca*~) required for nearly complete inhibition of arginine uptake depend on the amount of nuclear protein used, but usually 0.5 to 1.0 mM Ca ÷÷ and 1.0 to 3.0 mM E G T A are adequate. Although the statistical significance is questionable, it was often found t h a t K + produced better stimulation of arginine u p t a k e than Na* under otherwise identical conditions. For example, in Figure 1 at 10 minutes, N a ÷ = 35.5 -+ 2.7 and K* = 42.5 ~- 5.3. These results m a y depend on the variability in Ca ++ concentration from one experiment to another. I t can be shown with additions of progressively higher concentrations of Ca ++ t h a t N a + stimulation of amino acid uptake is more sensitive to inhibition than is K * stimulation. I t appears t h a t in the 10-~ to 10.4 M range of Ca ~, which is required for maximal activity of the amino acidstimulated adenosine triphosphatase, ~ K ~ is a better stimulator of amino acid uptake than is N a ÷ (see T a ble I I I ) . Nuclear uptake of serine: The uptake by heart nuclei of F~4C] i - ~ serine is less than the uptake of arginine or alanine (Table I). There is a definite preference for K ÷ over Na + to stimulate amino acid uptake, 5.3 and 2.5 times the estimated passive equilibrium value, respectively. The addition of 0.025 mM Ca +÷ has no significant effect. However, 0.05 ml of supernatant from the original myocardial homogenate inhibits uptake completely. This experiment was initially intended to test if some essential factor might be lost during nuclear isolation, which when added back would stimulate serine uptake.

Nuclear uptake of dicarboxylic amino acids: Conditions permitting alanine or arginine uptake produced no significant active or passive uptake of either [14C] L-a glutamic or aspartic acids, despite additions of various concentrations and combinations of Ca --, Na +, K ~ and pyridoxal.

Arginine uptake: competition by D-arginine, L-glutamic acid and L-alanine: To learn more about the transport of L-arginine, the possible competition b y its stereoisomer and by two other amino acids was investigated (Table I I I . The addition of unlabeled L-arginine up to 12 mM does not appreciably affect the u p t a k e achieved with 3 mM of L-arginine (data not presented). Exposure of nuclei to D-arginine causes a significant~enhancement of isotope uptake, whether the agent is added prior to or simultaneously with the [~4C] L-a arginine. The stimulated uptake is not appreciably affected by changing the ratio of D- to L-arginine from 1 : 1 to 4 : 1. I n the presence of 3 or 12 mM L-glutamic acid. which is not transported by the nuclei, ~here is no effect on L-arginine uptake. L-arginine is likewise un-

302

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TIME AT 30°(rain.) Net uptake of [14C]L-~ arginine (3.0 mM) versus t i m e a t 30C. o _~ basic Mg-- medium; contains 2.5 mM Tris-ATP, 2.5 mM MgCI,, 20 mM Tris-CI buffer at pH 7.5 and 260 mM sucrose to achieve approximate isotonicity. ~ ___ Mg ÷~ -I- Na ÷ or K÷ medium; contains the above plus 130 mM NaCI o r KCI and no sucrose. An average value from 52 exp e r i m e n t s for 10 minutes, including 41 with NaCI and 21 with KCI. Values at 5 and 3 0 minutes are averaged from 5 experiments, and the data are normalized to an average of 52 experiments as previously described. 1 Horizontal dashed line indicates theoretical passive equilibrium value for isotope. Vertical bars indicate ± 1 SEM. Figure 1.

affected in the presence of 3 or 12 mM L-alanine, which is transported by the nuclei.

Existence of arginine in an intranuclear diffusible pool: Previous evidence t h a t amino acids accumulated by the nuclei were freely diffusible I was mostly indirect. Therefore, experiments to analyze nuclei directly for possible incorporation into protein were performed. A typical u p t a k e of arginine was achieved m these experiments (Table I I I ) , but only 2.0 percent. of the label could be recovered in the acid and alkaline precipitable fractions, both values being of questionable significance.

Cation content of mid-fetal embryonic heart and isolated nuclei: The compelling in vitro evidence for the existence of an amino acid transport system in embryonic heart cell nuclei confronts one with the problem of how to correlate such biochemical data with conditions in the living cell. One would like to know exactly where the transport adenosine triphosphatase is located, and which c o m p a r t m e n t or compartments of the cell contribute the ]ons required for its activity and for optimal amino acid transport. I t was decided t h a t a combined approach, utilizing direct atomic absorption analyses of hearts and isolated nuclei together with a histochemical method ( K - p y roantimonate) and electron microscopy, would yield

The American Journal of CARDIOLOGY

NUCLEAR AMINO ACIDS AND CATIONS

TABLE I [14C]L-Serine Uptake (103 Counts/mg Protein/min) Experiments (no.)

Additions

Mg ++

Mg ++ + Na + Mg ++ -t- K+

--

22

1.7 ± 1.9

5.6 ± 1.7

12.0 ± 1.4

0.025 mM Ca ++

11

1.6 ± 2.2

4.9 ± 2,1

13.3 ± 1.9

4

1.2 ± 2.2

Supernatant

1.0 ± 2.3

Serine 3.0 raM; basic Mg ++ and Mg ++ + Na + or K + media as in legend for Figure 1. Supernatant, 0.05 ml from first 500 g centrifugation in nuclear isolation procedure. ~ indicates 1 SEM.

TABLE II []4C] L-Arginine Uptake (103 Counts/rag Protein/rain) Conditions Additions

-Pre-inc. 10 min

Mg ++

Mg-H-+Na +

1.5±1.1

-D-Arg: 3rnM

16

37.9±2.6 45.2±3.7

12mM

16

50.1±4.5

D-Arg: 3mM 12ram L-Glut: 3mM

16 16 6

48.9±2.9 50.5±3.8 33.4±6.8

12raM L-AM: 3raM

6 6

36.3±6.5 32.9±7.7

12raM

6

32.7±5.2

Average Overall

48,2±1.8 0-time 0-time

34,9±3.3

32.8±3.4

Arginine 3.0 mM; basic MR"H- and MR'H-+ Na+ media as in legend for Figure 1. "Conditions" column refers to time of addition of possible competitors relative to the addition of [14C] L-~ arginine. ± indicates I SEM,

TABLE III Arginine in an Intranuclear Diffusible Pool [14C]L-~ Arginine Uptake (103 Counts/mg Protein/rain) Experiments (no.)

Mg ++

8

1.4 ±2.6

Mg ++ + Na + Mg ++ + K+ 24.5 ±5.9

34.5 ±5.4

TCA Ppt

NH3 Ppt

0.23 0.24 ±0.05 ±0.08

Arginine 3.0 mM; basic Mg ++ and Mg ++ + Na + or K + media as in legend for Figure 1, except supplemented with 0.025 mM Ca ++. Values for acid and alkaline precipitates are the relative number of counts found in these fractions taken from an equivalent number of nuclei used in the Mg ++ + Na + or K+ medium; ± indicates 1 SEM. Ppt = precipitate.

VOLUME 25, MARCH 1970

the most informative results. Direct analysis is quantitative but has the drawback of nuclei in an unnatural habitat. The semiquantitative histochemical method m a y give a truer picture of the relative subcellular distribution of certain ions, but one needs to be cognizant of possible artifacts. From previous flux experiments 6 it is known t h a t at 12 days the embryonic heart is able to exchange a relatively high percentage of its K ÷ content, and the latter is present at 88 m ~ , ~,6 a concentration close to t h a t expected from recorded t r a n s m e m b r a n e potentials7 N a ÷ content is 48 mM, and only 30 percent of it is exchangeable. On the basis of the latter, together with values for the cation content of the various embryonic fluidsl 5 to 10 mM K + and 100 to 130 m ~ Na*, s it is presumed t h a t under normal conditions the m y o cardial cell nuclei m a y be exposed to two possible concentration ratios of N a + and K *. These are an extracellular environment of about 5 mM K ÷ and 125 mM Na*, and a cytoplasmic matrix environment of 15 mM Na* (free) and 115 mM K ÷. The latter K ÷ concentration is increased to give a total of 130 mM monovalent cation as used in studies of adenosine triphosphatase and amino acid uptake. Either Na ÷ or K ÷ at 80 to 130 mM will maximally stimulate the uptake of a d e n o s i n e triphosphatase a n d amino acid. Cation contents are given in Table IV. Ca*+ content: Freshly isolated nuclei suspended in 0.2 M sucrose contain 8.0 mM Ca *+ despite h a v i n g been exposed to 3.0 mM E G T A during the last purification step and suspension in a sucrose medium containing no more than 0.01 mM Ca ** as contaminant. T h e various combinations of N a ÷, K +, A T P and arginine have little effect on Ca ÷+ content. Nuclear Ca** exceeds t h a t in the myocardium. M g " content: Freshly isolated nuclei contain no measurable Mg** under present conditions, when suspended in a sucrose solution containing no more t h a n 0.001 mM Mg ÷÷ as contaminant. The concentration of Mg *÷ is 5 to 7 times t h a t in the nuclear suspending medium with 2.5 mM added. When A T P is present the M g ÷÷ content is considerably reduced in the nuclei. Na* content: Freshly isolated nuclei contain about 28 mM Na+ despite isolation in Na*-free media and suspension in a sucrose solution containing no more than 0.05 mM Na * as contaminant. The increases in nuclear N a + content in low Na*-high K s and in high Na*-low K ÷ media reflect reasonably well t h a t additional amount of Na + added to the suspending medium. The addition of A T F and arginine has little effect. K + con.tent: Freshly isolated nuclei contain little if any K *. In a high Na+-low K ÷ medium, nuclei eoncent r a t e K ÷ about 3 times. In low Na+-high K+ medium, the nuclear concentration is 40 to 50 percent above t h a t in the environment. A T P and arginine appear to have little effect.

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KLEIN ET AL.

TABLE IV Twelve.Day Embryonic Heart Cation Content Conditions

CA ++ A.

Range

Mg ++

Range

Na +

Range

K+

Range

Nuclear Cation Content (millimoles/liter)

0.2 M sucrose

8.0

2-20

0

28

6-39

1.1

0-6.5

125 mM Na + - 5 mM K+

8.6

3-15

16.9

6-30

188

138-276

16

6-33

above plus 2.5 mM ATP, 3.0 mM Arg.

7.8

2-19

7.0

0-14

168

105-324

15

15 BM Na + --115 mM K+

5.8

2-11

12.6

3-24

39

21-62

164

86-246

6.4

3-11

2.7

0-10

45

29-63

168

112-290

above plus 2.5 mM ATP, 3.0 mM Arg. 0.2 M sucrose (no tissue)

0.01

B. Normal Ringer's

0.001

0.05

7-27

0

Ventricle Cation Content (millimoles/kg wet) 1.7

1.4-2.1

6.6

5-8

48

30-63

88

78-96

Nuclei: isolated and incubated in above media buffered to pH 7.5 with 20 mM Tris-CI for 15 minutes at 30C. All media except sucrose contained 0.025 mM Ca++ and 2.5 mM Mg ++, which are optimal for amino acid transport and adenosine triphosphatase activity. An average of 8 experiments with all cations and media in each. Ventricle: equilibrated in normal Ringer's medium for 30 minutes to 1 hour. Ca ++ and Mg ÷+ values an average of 13 hearts; Na + and K+ values from previous data. 5,6 Atomic absorption data on Na + and K+ agreed well with the latter.

Histochemical

l o c a l i z a t i o n o f N a + a n d Ca ++ p l u s

Mg++: The K-pyroantimonate method for Na + and Ca ++ plus Mg +÷ gives some information as to the intracellular localization of these cations. Also a semiquantitative estimate of the relative concentration differences at various subcellular sites can be made by determining the number and size of precipitate particles per unit area (see Methods). Experiments were performed on whole hearts or isolated right ventricular strips depending on embryonic age. Present data are limited to findings at mid-fetal age (10 to 13 days), which are pertinent to the amino acid transport data. Series of hearts were fixed immediately after excision in Os04 with and without Kpyroantimonate. Other series were pre-equilibrated in oxygenated Ringer's medium or Ca++-free Ringer's medium plus 1.0 or 3.0 mM E G T A prior to fixation. In normal Ringer's medium, hearts resume rhythmic contractions and reach a steady state of cation equilibrium. ~,6 In EGTA-Ringer's medium, hearts quickly become quiescent. K-pyroantimonate-treated tissues should demonstrate Na*, Ca ++ and Mg +÷, whereas tissues pretreated with excess E G T A should demonstrate primarily N a +, indicating indirectly where Ca ~* and Mg +÷ have been removed. Initially there was some uncertainty about Mg-antimonate precipitating under these conditions, as suggested by Komnick and Komnick. '9 Therefore, a series of Mg *+ concentrations between 1.0 and 0.01 mM in cold 0sO~-K pyroantimonate fixative were tested and found to produce turbidity, which could be measured spectrophotometrically even at the lowest concentration. It is clear that our conditions should indicate Mg +÷ with the large excess of antimonate present.

304

It can be calculated t h a t the concentration of E G T A used is also in large excess of the Ca +* plus Mg ++ present in the tissue; thus, both should be chelated and washed out. Ringer's medium contains no Mg +*.

A typical section ]rom right ventricle of the 12-day embryo heart fixed immediately upon excision is shown (Fig. 2) for comparison with one from a right ventricle pre-equilibrated in oxygenated Ringer's medium for 30 minutes at room temperature before fixation (Fig. 3). Neither control sections nor antimonate-treated sections were stained to give maximal contrast. Distribution of precipitate in tissues fixed immediately in O s Q - K pyroantimonate does not differ appreciably from those pre-equilibrated in Ringer's medium (Table V) and is not illustrated.

An antimonate-~reated preparation which was preequilibrated in Ringer's medium is shown in Figure 4, and one from the same experiment pre-equilibrated in Ca++-free Ringer's medium plus E G T A in Figure 5. Precipitate is localized heavily in nuclei and occurs mostly in nucleoli, chromatin areas and along the inner nuclear membrane. I t is less concentrated in the remaining nuclear matrix and is absent from the immediate area of the pores and perinuclear space. In the myofibrils, precipitate is found in relatively high concentration close to and paralleling the Z lines and scattered less densely throughout the A band. In our experiments, cold 0 s Q - K pyroantimonate fixative produces contracted myofibrils obliterating the I band, whereas they are relatively more relaxed in preparations pretreated with EGTA. Precipitate is lightly scattered throughout the cytoplasmic matrix, mitochondria, vesicular elements of endoplasmic retieulum and Golgi. None is found in desmosomes or

The American Journal of CARDIOLOGY

NUCLEAR AMINO ACIDS AND CATIONS

Figure 2. Section from control 12 day embryonic right ventricle fixed immediately after excision in cold acetatebuffered Os04. N ---- nucleus. ( X 18,000, reduced by 10 percent.)

in elements forming intercalated discs. Relatively dense precipitate borders lipid droplets but disappears if the lipid is extracted by fixation and embedding procedures. Little if any precipitate is found in areas of glycogen accumulation, between parallel plasmalemmae of adjacent cells or in lacunae between cells. Only a small amount is adsorbed to the plasmalemmae. In EGTA-treated preparations there is an obvious difference in the relative distribution of antimonate precipitate, presumably due to removal of Ca +* and M g ' . There is some reduction in nuclear precipitate, but it appears to occur primarily in the nuclear ma-

trix, as Opposed to nucleoli, ehromatin and along the inner nuclear membrane. There is a striking decrease in precipitate paralleling the Z lines and in the A band; compare control medium (Fig. 6), normal Ringer's medium (Fig. 7) and Ca÷+-free Ringer's medium plus EGTA (Fig. 8). The precipitate scattered in the cytoplasmic matrix and mitochondria is reduced, but that at the surface of lipid droplets appears unchanged. Semiquantitative estimate of relative amounts of antimonate precipitate: The amounts of antimonate precipitate at various subcellular loci are estimated (see Methods), and that in the cytoplasmic

Figure 3. Section from control 12 day embryonic right ventricle preequilibrated in oxygenated Ringer's medium at room temperature for 30 minutes before fixation in cold acetatebuffered OsO~. N -- nucleus; G = Golgi; L ----- lipid. (X 24,000, reduced by 10 percent.) Inset is a cross section of nuclear pores showing octagonal symmetry. ( X 54,000, reduced by 10 percent.)

VOLUME 25, MARCH 1970

305

KLEIN ET AL. TABLE V Estimate ef Relative Amount of Antimonate Precipitate at Subcellular Sites Micrographs Counted

Cytoplasmic Matrix

Conditions

Nucleus

Mitochondria

18

No pre-equilibrium

46

Ringer's

5.73 (4.7-7.4) 5.16 (3.7-7.2)

1.1 (1,0-1.3) 1.0

1.0 (0.7-1.0) 0.91 (0.9-0.95)

55

Ca++ - free Ringer's + EGTA

3.90 (3.5-4.2)

0.39 (0.32-0.45)

0.25 (0.23-0.27)

Z Line

A Band

2.15 (1.9-2.3) 2.29 (1.8-3.2)

1.39 (1.1-1.6) 1.32 (1.1-1.5)

0.49 (0.47--0.51)

0.37 (0.35-0.39)

Ranges in parentheses are of the means of the counts by 4 individuals. See Methods for counting procedure.

matrix is arbitrarily set equal to one in preparations pre-equilibrated in normal Ringer's medium (Table V). Values for myocardium that has been OsO4-K pyroantimonate-fixed directly from the embryo and that which has been pre-equilibrated in normal Ringer's medium should indicate Na ÷, Ca ++ and Mg ~÷ distribution. Most precipitate is found in the nucleus and is about 5 to 6 times that in the cytoplasmic matrix. No attempt has been made to differentiate between intranuclear areas of heavy and light precipitate. A random sampling of the whole nucleus was taken and averaged: The immediate area paralleling and including the Z lines has precipitate concentrated at least 2 times that in the cytoplasmic matrix, while that in the A band is about 1.3 times. The antimonate precipitate should indicate primarily Na ÷ distribution in myocardium pre-treated with Ca*+-free Ringer's plus EGTA. The ratio of precipitate in the nucleus compared to that in the cytoplasmic matrix is increased to 10:1. There is approximately 24 percent reduction in intranuclear precipitate due to C a +* and Mg ++ removal. There is a 60 percent reduction of the randomly scattered precipitate in the cytoplasmic matrix. Thus, about 40 percent of the original antimonate precipitate in tissues without EGTA represents Na*. Precipitates in the Z line area, the A band and mitochondria are reduced 70 to 75 percent by treatment with EGTA. A comparison of antimonate distribution with lead salt indicative of presumptive ATP hydrolysis: It is of interest to compare the distribution of Na-pyroantimonate precipitate with that of lead salt resulting from ATP hydrolysis,1° even though the latter must be viewed with qualified acceptance because of possible artifacts. A section from a 12 day embryonic heart is shown in Figure 9. Lead salt is found paralleling the inner nuclear membrane, in the nucleolus and surrounding the nuclear pores. The antimonate precipitate is not found at nuclea~ pores but does occur along the inner nuclear membrane and in nucleoli. The localization of adenosine triphosphatase at the nuclear surface is supported by evidence from our

306

own data, 1,3,~°,11 other histochemical studies 12 and biochemical studies with isolated nuclear membranes?3

Discussion Amino acid transport: It can be concluded that isolated 12 day embryonic heart nuclei are capable of transporting a number of amino acids, which are held in a concentrated intranuclear pool and in freely diffusible form. The present data eliminate the possibility of any appreciable incorporation into protein but do not rule out incorporation of smaller peptides. The latter possibility is being checked by chromatography. Under optimal conditions, the efficiency of nuclear transport differs for the three amino acids; arginine ,~ 2X > alanine -~ 1.3X > serine. There is no competition between L- and D- arginine. In fact, under conditions in which L-arginine transport sites appear to be saturated (maximal isotope uptake and ATP hydrolysis), the uptake of L-arginine is stimulated in the presence of the D-isomer, but not by L-alanine or L-glutamic acid. One explanation is that the D-isomer enters the nucleus and an exchange diffusion follows between stereoisomers, but not between unlike amino acids. L-and D-alanine are equally well transported by the nuclei. 1 Neither L-glutamic nor L-aspartic acid is accumulated by the nuclei. There is considerable evidence14 that dicarboxylic acids do not permeate isolated nuclei. Embryonic heart nuclei differ from calf thymus nuclei2 by not being stereoisomer-specific for alanine and by alanine being accumulated in the presence of either Na ÷ of K+; thymus nuclei are specific for the L-form and utilize only Na + for alanine. However, in many respects the two systems are similar: They both require a monovalent cation, Mg ÷÷and ATP; they are selective for certain amino acids; competition does not occur between unlike amino acids; they are temperature-sensitive; and amino acids are not held for long periods in the intranuclear pool. Nuclear and myocardial cation contents: Isolated nuclei from the 12 day embryonic heart have

The American Journal of CARDIOLOGY

NUCLEAR AMINO ACIDS AND CATIONS

Figure 4. Section from experimental 12 day embryonic right ventricle preequilibrated in oxygenated Ringer's medium at room temperature for 30 minutes before fixation in cold acetatebuffered OsO,-K pyroantimonate. D --disc; GI ---- glycogen (partly extracted); N ---- nucleus. ( × 18,000, reduced by 10 percent.)

the ability to bind Ca *+ (8 raM) in an environment containing 10-5 M or less of this cation. In 13 d a y chick embryonic skeletal muscle, 65 percent of the tissue Ca +* was reported to be intranuclear? ~ Calculations based on a nuclear content of 8 mM Ca ÷÷, a m y o cardial content of 1.7 mM Ca ÷÷ and a nuclear:cytoplasmic ratio of 1:9 suggest t h a t there is 1 mM Ca÷~ in the extranuclear cytoplasm and 40 to 45 percent of the total Ca*+ in the nucleus of 12 d a y embryonic heart. The ability of the nucleus to retain considerable amounts of Ca ÷* even after E G T A t r e a t m e n t lends credence to our earlier speculation t h a t a Ca *÷ inactivating mechanism could govern the rate of energy

release from A T P available for use in amino acid transport. 1~ An environmental Ca ÷÷ between 10-5 and 10-4 M produces optimal adenosine triphosphatase activity and amino acid transport; both are inhibited below and above this range. 1~ Embryonic heart nuclei also have the ability to bind N a ÷ (28 raM) in an environment of 10-5 M of this cation. A similar level of bound N a ÷ was demonstrated in t h y m u s nuclei. 1~ In addition to bound N a ÷, this cation is concentrated by heart nuclei at a level reasonably paralleling t h a t in the suspending medium and probably reflects t h a t concentration in the extracellular environment. The latter is suggested by the present N a ÷-

Figure 5. Section from experimental 12.day embryonic right ventricle preequilibrated in oxygenated Ca++.free Ringer's medium plus 3.0 mM EGTA at room temperature for 30 minutes before washing and fixation in cold acetate-buffered OsO~-K pyroantimonate. D = disc; L = lipid; N -~ nucleus. ( × 18,000, reduced by 10 percent.)

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KLE!NET AL,

Figure 6. Control sarcomere as in Figure 3. Z ~- Z line. ( × 54,000, reduced by 10 percent.)

Figure 7. Sarcomere, Ringer's medium as in Figure 4. Z = Z line. ( X 54,000, reduced by 10 percent.)

Figure 8. Sarcomere, Ca*÷.free Ring. er's medium plus EGTA a s in Figure 5. Z ~ Z line. ( × 54,000, reduced by 10 percent,)

Figure 9. Section from 12 day embry. on ic right ventricle reacted with modified Wachstein and Meisel medium after glutaraldehyde fixation.,,Post-fixed in Os04, stained with uranyl acetate. L ----- hpld; N ~_ nucleus. Small arrows _-- nuclear pores; large arrow ~- lead precipitate between adjacent plasmalemmae; (X 15,000, reduced by 10 percent.)

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NUCLEAR AMINO ACIDS AND CATIONS

p y r o a n t i m o n a t e distribution studies I see later), and was previously shown for other nuclei using nonaqueous isolation techniques, ls-19 M g - is not tightly bound by the nuclei but can be concentrated 5 to 7 times the 2.5 mM found optimal for the adenosine triphosphatase, which is also typical of M g - concentrations found in the serum and extraembryonic fluids at 12 days. s The marked reduction of nuclear M g ** in the presence of 2.5 mM A T P ( I : I with Mg ~+) suggests t h a t A T P chelation of Mg ÷* is highly competitive with nuclear binding sites and t h a t the nuclear membrane is relatively impermeable to the M g - A T P complex. Nuclear impermeability to A T P has been reported in other tissues ~4 K ÷ is not tightly bound by the heart nuclei. This is also true for thynms nuclei. ~7 The intranuclear K ÷ in heart reflects t h a t found in the suspending medium except t h a t it is concentrated 3 times at 5 mM and 1.4 times at 125 mM extranuclear K *. The latter is in agreement with work on liver nuclei. 19 I t is tempting to suggest that the tuner nuclear membrane is selective for Na* via the perinuclear space, but this cation can not leak into the cytoplasmic matrix through the nuclear pores. Further. that the nuclear pores are selective for K *. and this cation does not leak out through the inner nuclear merebrahe. Thus. free Na ÷ in the nucleus could be in equilibrium with extracellular Na ÷. and only a concentration gradient for K ÷ m a y exist. If the intranuclear and cytoplasmic matrix K ~ are entirely ionized at a ratio of 1.4: 1. respectively, it can be calculated using the Nernst equation, t h a t the nuclear t r a n s m e m brane potential is about 9 my. This value is reasonable. judging from microelectrode studies on other nuclei. 2° Histochemical

localization of cations:

Based on

antimonate precipitate distribution after E G T A treatment. ~he nucleus contains about 10 times as much N a ÷ as does the cytoplasmic matrix. This ]s in agreement with data on liver nuclei, ~s,~9 Using a concentration of 15 mM free N a ÷, as determined by isotope exchange in this tissue at 12 days, 6 the nucleus should contain about 150 mM Na +. This is r e m a r k a b l y close to the values by direct analysis of 168 to 188 mM N a *. Using an average of the latter and 15 mM Na + in the cytoplasmic matrix, it can be calculated t h a t the nucleus would have to occupy 20 percent of the total cell volume to account for the 48 mM Na+ in the 12-day embryonic ventricle. Judging from low power electron micrographs, the average diameters of myocardial cells compared to nuclei fall between 2:1 and 3:1 at this age, giving a value of 4 to 12 percent for the volume of the cell occupied by the nucleus. Thus. at most, the nucleus can account for 50 to 60 percent of the non-

exchangeable Na ÷ at this age. T h e additional bound N a ÷ could be in the cytoplasm. Other studies using p y r o a n t i m o n a t e suggest t h a t precipitate in this phase does decrease with age. 21 Our earlier data show t h a t after the establishment of an effective N a - p u m p at 4 to 7 days of embryonic age, 22 the ventricUlar Na* content decreases from 48 mM at 12 days to about 30 to 35 mM after hatching, with a corresponding increase in N a ÷ exchangeability from 30 to 73 percent. This was interpreted as a shift in equilibrium between free and bound Na ÷ with a consequent net loss of tissue N a ~ from the bound phase. 22 Our results on embryonic myocardial distribution of antimonate precipitate are in general agreement with the brief description by Yeh and Hoffman. 21 except for their "note in added proof." The present data m a y be compared in more detail with the recent study by Legato and Langer 2~ employing dog papillary muscle. We find very little precipitate in the extracellular spaces between closely parallel plasmalemmae, in lacunae or adsorbed to the plasmalemmae, in contrast to the h e a v y precipitate reported for papillary muscle. As all of our antimonate-treated preparations were relatively contracted, the sarcomere precipitate was always found immediately adjacent to the Z lines. In E G T A - t r e a t e d preparations, which were more relaxed, remaining precipitate still appeared adjacent to the Z lines rather than in the center of the I band as foUnd in papillary muscle. However, I band localization w a s difficult to judge in our material because antimonate was much less densely deposited than in papillary muscle and more completely removed by E G T A . This could also explain our inability to conclude t h a t a lesser precipitate was found in the M band region t h a n in the rest of the A band as found in papillary muscle. Although the probability m a y exist, we hesitate to state t h a t the myofibrillar precipitate removed by E G T A represents primarily Ca +÷, One is obliged to accept t h a t E G T A in large excess of tissue Ca *÷ will also chelate and remove M g " , on the basis of reported association constants. ~4 It cannot be claimed t h a t the loss of precipitate from a subcellular locus after E G T A t r e a t m e n t is due primarily to C a " removal, unless the ligand can be shown to be present at a concentration which will just remove t h a t amount of Ca *+ found in the tissue. The latter situation will take maximal advantage of the superior chelating ability of E G T A for Ca ÷÷ compared to M g " . In the present experiments it is untenable to suggest t h a t there is one and a half times as much Ca *+ as there is N a ÷ in the cytoplasmic matrix, which would be indicated by E G T A studies if one assumed only Ca** was removed. Direct Cation analysis does not substantiate this.

References 1. Klein RL, Horton CR, Thureson-Klein A: Evidence for an amino acid transport system in nuclei isolated from embryonic heart, Europ J Biochem 6:514-524, 1968

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2. AIIfrey VG, Meudt R, Hopkins JW et al: Sodiumdependent "transport" reactiohs in the cell nucMus and their role in protein and nucleic acid synthesis. Proc Nat Acad Sci USA 47:907-932, 1961

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KLEIN ET AL. 3. Klein RL: A histochemical and biochemical study of nuclear adenosine triphosphate hydrolysis in embryo heart. J Histochem Cytochem 14:669-680, 1966 4. Komnick H= Electronenmikrokopische Lokalisation von Na÷ und CI- in Zellen und Geweben. Protoplasma 55:414-418, 1962 5. Klein RL, Evans ML= Effects of ouabain, hypothermia and anoxia on cation fluxes in embryonic chick heart. Amer J Physiol 200:735-740, 1961 6. Klein RL" Ontogenesis of K and Na fluxes in embryonic chick heart; Amer J Physiol 199:613-618, 1960 7. Yeh BK, Hoffman BF" Ionic basis of electrical activity in embryonic cardiac muscle. J Gen Physiol 52:666-681, 1968 8. Romanoff AL" Biochemistry of the Avian Embryo. New York, John Wiley, 1967, p 140-141, 168-169, 172-173 9. Komnick H, Komnick U= Electronenmikroskopische Untersuchungen zur funktionellen Morphologie des Ionentransportes in der SalzdrOse von Larus argentatus. Z Zellfo rsh 60:163-203, 1963 10. Klein RL, Afzelius BA: Nuclear membrane hydrolysis of adenosine triphosphate. Nature (London) 212:609, 1966 11. Klein RL: ATP hydrolysis by isolated embryonic heart nuclei using a histochemical method. Proc Soc Exp Biol Med 124:1258-1260, 1967 12. Yasuzumi G, Tsubo I: The fine structure of nuclei as revealed by electron microscopy. II1. Adenosine triphosphatase activity in the pores of nuclear envelope of mouse choroid plexus epithelial cells. Exp Cell Res 43:281-292. 1966 13. Zbarsky IB, PerevoshChikova KA, Delektorskaya LN, et al" Isolation and biochemical characteristics of the nuclear envele pc. Nature (London) 221:257-259, 1969

14. Georgiev GP: The nucleus, Enzyme Cytology (Roodyn, DB, ed). New York, Academic Press, 1967, p 81, 85 15. Cosmos E: Intracellular distribution of calcium in developing breast muscle of normal and dystrophic chickens. J Cell Biol 23:241-252, 1964 16. Klein RL: Ca** requirement for Na + stimulated Mg-ATPase in nuclei isolated from embryonic heart. Exp Cell Res 49:69-78, 1968 17. Itoh S, Schwartz IL: Sodium and potassium distribution in isolated thymus nuclei. Amer J Physiol 188:490-498, 1957 18. Langendorf H, Siebert G, Nitz-Litzow D: Participation of rat liver nuclei in movements of sodium. Nature (London) 204:888, 1964 19. Siebert G, Humphrey GB: Enzymology of the nucleus. Adv Enzymol 27:239-288, 1965 20. Loewenstein WR, Kanno Y: Some electrical properties of a nuclear membrane examined with a microelectrode. J Gen Physiol 46:1123-1140, 1963 21. Yeh BK, Hoffman BF: A hiStochemical and electrophysiological study of chick heart embryogenesis: localization and comparison of myocardial sodium content and the mechanism of excitation, Myocardial Contractility (Tanz RD, Kavaler F, Roberts J, ed). New York, Academic Press, 1967, p 279-291 22. Klein RL: The induction of a transfer adenosine triphosphate phosphohydrolase in embryonic chick heart. Biochim Biophys Acta 73:488-498, 1963 23. Legato M J, Langer GA: The subcellular localization of calcium ion in mammalian myocardium. J Cell Biol 41:401-423, 1969 24. Bjerrum J, Schwarzenbach G, Sill6n LG: Stability Constants. I: Organic Ligands. London, The Chemical Society, 1957, p 76, 90

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