Ionic composition of the rat aorta after incubation in saline

Vol, a, Part I, pp. 919-927, 1989. ~e gc~entes Printed in Great Britain

Pergamon Press

IONIC COMPOSITION OF THE RAT AORTA AFTER INCUBATION IN SALINE 6eorges Roriv~ Department of Biochemistry (Prof .M . Florkin) and Department of Medical Clinic and Pathology (Prof . H . Van Cauwenberge) University of Liège,

Belgium

(Received 5 Ma,y 1969; in final form 9 June 1989)

IT has been repeatedly reported that during incubation is saline., smooth muscle cells take up sodium and lose potassium. (1-12) .

Usually, the modification described is partly reversible, but the ionic composition of the smooth muscle in vitro ~r~p~y~as different from that in vivo .

The study of the distribution of ions between the different compartments of a tissue, and of the ionic shifts accompnnying

the smooth muscle contraction, are usually performed in saline . Thus, the modification of the ionic composition when the smooth muscle is transferred to saline must be known, and, if possible,

prevented .

We have therefore studied the ionic shift during the four first hours of the incubation of the rat aorta in saline ae troll as the effects of various experimental conditions such as oxygenation, anoxia and ouabain .

Material and Methods Aortas from White Star fema]e rata weighing 200-250 g are analysed for sodium, potassium and total water before or after

incubation in saline . The rat is killed by a blow on the skull and bled immediately . The aorta is rapidly and very carefully diPaected free of connective tissue, in situ , with micrôdiseection

instruments . The aorta is then sectioned on the ti~oracic arch and just below the renal arteries . After removal, the aorta is alit open lengthwise . All the dissection procedure requires four to six min~Ytea .

Ten aortas are gently blotted on filter paper (Selects 589) to remove moat of the adherent blood . The tiaeue ie put into a tared weighing bottle . The wet weight (WW), the dry weight (DW) ~Äspirant du Fonda National de la Recherche Scientifique

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and the amount of sodium and potassium are measured as described in a previous paper (13) . The other aortas are incubated in a saline at 37' C having the following composition in mSq/L : Na 142,

ä 5 " 3, C1 127, Ca 2 .6, Mg 2 .4, H 2P0 4 2 .4, S0 4 2 .4, and pH 7 .4 .

Four experimental conditions are investigated a) Zn this group, the saline is oxygenated by gently bubbling 02 (5-10 ml 02/min/ml saline) . b)

In other experiments (high oxygen group), the oxygen flow ie increased to 30-$0 ml 02/min/ml saline) .

c) To study the effects of anoxia, oxygen is replaced by nitrogen . d) Finally, in the last experimental group, the aortas are incu4 M. bated in an oxygenated saline containing ouabain 10 After incubation, the preparation is gently blotted on fi]ter paper and WW  DW and amount of sodium and potassium are measured as described below .

Reaulta A . Ionic comDOaition of the rat aorta after incubation in saline Fig . 1 shows the variations of total water, total sodium

and potassium of the rat aorta during the'firat minutes of incubation in an oxygenated saline (group a) . After 15 sec . of incubation, th9 sodium content of the preparation increases from 71 .56± 3 .2 mEq Na/kg WW to 90 .80±2,7 mEq/kg WW . This uptake of sodium is

maximum after 15 sec . of incubation . The concentration of thin cation then decreases progressively until a value as low ae 74.42

±1 .38 mTq/kg WW is reached after 30 min . The total sodium of aortas incubated for a longer time (60, 120 and 140 min .) is not significantly higher than that of aortas incubated for 30 min . but

remains higher (P ~ 0 .001) than the sodium content of uniacubated aortas . When the sodium content of the aorta is expressed as m$q/kg DW, the variations recorded during the .firat minutes of the incubation in saline are similar to those described above, but are generally higher because of variations of the total water of the incubated aortas . After 15 ~ec . of inoubation, the_potaasiusi

content decreasea .This apparent shift in total potassium is probably related to the uptake of water by the ports, because if the

results are expressed in terms of DW, the a~sount of potassiumi found for the incubated aortas (lOZ,63±3 "32 mg4~g DW) is the same as the control values

(101 .68±4 .56) . (Fig . 3) .

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After a longer incubation in saline, total potassium of rat

aorta decreases progressively to the lowest value after 5 and 10 min . of incubation . These values are significantly lower than t1iH control whether the results are expressed in relation to the WW or to the DW of the preparations . After 15 min . of incubation,

the total potassium has always the same value as that of tt~e con-

trol . From 30 to 240 min . of incubation no more important changes can be detected . However, it must be pointed out that the total potassium of incubated aortas remains always lower than the control v»lues . The modifications of total water in incubated preparations

are parallel to that of sodium . During the first few seconds of incubation, aortas take up water (653±26 .7 ml H2 0/kg WW as compared to 599±27 .5 ml H2 O for control aortas) . Subsequently, vari ations of total water of the preparation are small, except again after 15 min . of incubation . At this time, water content is as great as 67$±13 .5 ml . On the contrary, the values measured after 20 or longer incubations are similar, but remain significantly Greater than the control values .

B . Effect of oxygenation,_ on the ionic modifications of the rat aorta_ during incubation in saline . Increasing the flow of oxygen five or six times has no measurable effect on the ionic modification described in the

first chapter (Fig . 2) . Only some isolated values differ from

those found for the preparations incubated in normal conditions

(group a) . for instance, after 20 min ., total sodium is lower in high oxygen, but at 60 min . i t is higher than in gentle bubbling . In the same way, the modifications of total water are similar to those recorded in the aortas incubated in normal conditions, except after 60 min . of incubation . At this time, total water is

notably higher than in the first experimental group : 660±12 ml H2O and 639±10 ml H20/kg WW . The variations of the potassium content of the aortas are the same in the low and high oxygen groups . No statistically significant differences can be found, except,

once again, after 15 min . At this time, the experimental values are lower in high oxygen (104 .37±6 .9 compared to 126 .07±5 .2 mEq K ~Icg DW in the fir at experimental group) .

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C . Effect of anoxia_ on the ion ç çomposition of the rat aorta

in vitro . During incubation in saline with nitrogen, the variations of total potassium are similar to those observed in the two first experimental groups (a and b) .Expreased in terms of mßq/kg DW, the potassium content of the preparation incubated in anaerobic

conditions are generally higher than control values . Hut the variations are not statistically different, except after 30 min .

(I~ig . 3) . On the contrary, the aortas incubated in saline with ni-

trogen bubbling take up more sodium than the aortas from the first experimental groups (a and b)

(Fig . 2) . This result is obtained

whether the sodium content.ia expressed in terms of WW or in terms of DW . During incubation in anoxic conditions, the preparations do not lose this excess of sodium as tion in the presence of oxygen .

occurs during incuba-

D . Effect of ouabaia The effect of ouabain (10- ~) on the ionic composition of the

rat aorta incubated in saline is dramatic .(Fig . 4) . The water content increaeea ae obaerve .d in the control group (a) during the first 20 min . of incubation . Hut later the total water continues to increase progressively in the preparations incubated with ouabain . However only a few discrepancies are measured (Fig . 4) . On the other hand, the sodium content greatly increaeea . Except

after $ min . of incubation, all the measured values, in this last experimental group (d) are significantly higher than in the con-

trol group(a), whether the reaulta be expressed in terms of WW or in terms of DW . It is worth noting the inflection of the curve after 1$ min .(Fig .4) . The potassium content is significantly decreased after incubation in saline containing ouabain 10 - ~,

except after 1$ min . of incubation . The ionic vaiationa measured are higher in this last experimental group than in the previous onee and are probably related to the pharmacological properties of the drug . Discussion Incubation in saline modifies the ionic composition of rat aorta . The modifications affect sodium, water and to a lesser extent potassium . However, the increase of total water and the decrease of total potassium are smaller than those described by

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other authors . For instance, Dawkins and Bohr

925 (4) note that, 4~fter

15 sec . of incubation in saline as well as is plasma, the potassium content of the rat aorta falls from 131 .1±1 .85 m8q/kg DW (aem) to values as low as 38 .0±4 .06 mEq/kg Di~I . Similar observations are reported by Bevan (1960) concerning the rabbit aorta,. However, this author urea a rough dissection procedure . Hy atn-

dying the rabbit aorta Dood and Daniel (5) find eoaller and not significant variations in the potassium content (from 27 .1 to 21 .1 mEq/kg WW) . In our work, the variations of potassium when expressed in terms of WW can partly be explained by the evaporation during the dissection procedure, and the uptake of water during the first minutes of incubation in saline . But the de-

crease of potassium observed for longer incubation periods, cannot be explained by variations in the water content . The increase in the amount of sodiua can only be explained by an uptake by the cells,

since the increase in sodium is higher than that of water .

Indeed, if the uptake of sodium was only or mainly located is the extracellular fluid, the increases in water and sodium should be

in the same proportion as that in the medium of incubation (145 mEq Na/L H20) . The rite of exchange seems to imply however that the function of the cell membrane is very strongly affected .

Similar observations have already been published by Daxkiaa and Bohr (4.) with rat aorta, and Goodford and Hermanaen (7) with the guinea pig Taenia coli . However, generally, the modification of the ionic composition of the artérial wall is related after 1, 2 or 4 hours of incubation, to prove the survival of the biological material and to control the stability of its ionic composition . Moat workers found no modification of the potassiua

content, between 1 and 4 hours of incubation, but a alight increase in total sodium, and thus an increase in the total catioas of the preparation . However, the new steady state obtained is vitro remains generally different from the ionic composition of the non-incubated preparations (2, 4, $, 12) . However, in a re-

cent work, Yillamil et al . (14) do not find substantial sodificationa is the sodium and potassium oonteat of the dog carotid artery incubated in saline . Hut these authors take as control pr~parationa an artery immersed several times is saline . Ia our ez-

perimeatal conditions, the modifications found between 10 sad 110 min . of incubation are also weak and non-significant (P ~ 0.10)

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The reaeonfor these modifications must be mainly looked for

tin the dissection trauma . Indeed if one perfusea rat aorta with saline in situ the modification of the ionic composition, mainly the sodium uptake, is less than during incubation in vitro in a

~Ame saline (4) . The dissection procedure can operate in two dif-

ferent ways . As shown in a previous publication (13), mishandling

of the preparation can irreversibly damage part of the smooth muscle cells . The cellular fluid of these muscular fibers equalizes rapidly with extracellular dodium . Such modifications should be

irreversible . This situation can probably explain the results of $evaa (2), Dawkina and Bohr (4) and Dood and Daniel (5) . It ;could also be argued that metabolism is altered in such a way as ~to reduce the amount of ATP available, thus leading to an important shift in the distribution of electrolytes . Our observations show that anoxia and ouabain amplify the

tonic modifications described during the earlier part of the incubation in saline, and delay the corrections of these ionic shifts . This constitutes an argument for the second explanation .

Hut these observations show also that during the first minutes of incubation the cells are able to control their ionic composition since anoxia amplifies uptake of sodium, and since ouabain amplifies both sodium increase and potassium decrease . ids may thus conclude that our dissection procedure gives a

viable preparation . During the first 20 min . of the incubation is saline, the water and cation content of the rat aorta modifies greatly . Rapidly these modifications are partly corrected so that the preparation may be regarded ae being in a new steady state nfter 20 min . of incubation till 120 and perhaps 240 min . of in-

cubation . However, the steady state obtained is vitro remains always different from that in vivo .It is iatereating to mention

that the increase in sodium is not necessarily coupled to the lose of potassium . In fact during incubation in oxygenated saline,

the uptake of sodium is more important than the decrease in potassium, and thus the sum of the cations barely increases whethi~r r6ssnhtab~ expressed in terms of mEq/kg WW (from 112 .91 to 121 .81 mSq/kg Wh after 5 min . for instance, or in terms of mEq/L H2O (191 .00 to 195 " 30 mEq/L H2O after 5 min~l.During anoxia too the movements of sodium are more important than those of potassium . In the saline-containing ouabain also, the increase in sodium is

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more rapid and trice as important ae the dacreaae in potassium .

This discrepancy betxeen the movements of the txo main catioae could partly be explained by variations is the extracellular apn~ ce . The determination of the volume of the extraoellular fluid, by measurement of the volume of diffusion of iaulin requires

at least 40 min . of incubation . Suoh a determination xaa there-

fore inapplicable in this study .

Sumo a~ 1 . Rnt aorta incubated is saline shorn, at the beginning of the

incubation, fir et an important increase in sodium, a loxer rise of total xater and a decrease in potassium, rhea compared with

normal values obtained for unincubated preparatioaa .After 20 min . of incubation, the sodiusa decreases but remains higher and the potassium loxer than the control values . 2 . High flox of oxygen does not prevent these ionic events .

3 . Anoxia increases still further the uptake of sodium, but does not modify the lose of potassium .

4 . Ouabain accelerates both the decrease in potassium sad the in-

crease in sodium content .

References l . L . ToBIAN and A . Fos, J . clia . Inveat .3~, 297 (1956) . 2 . J .A . HSVAN, J . pharmacol . ~cp .Therap . 129, 417 (1960) . www

(1960) 3 " S .S . DANIEL and S . ROBINSON, J . Physiol . (Londoa ),1~4,411 . . 4 . O . DAi~TSINS aad D .F .BOHR, Am . J . Phyaiol .~s~, 28 (1960) . 4$1 (1960) . 5 " W .A . DOOD and S .S . DANIELS, Circulation Ree .B, w 6 . C .Y . SAO, Am . J . Physiol . 201, 717 (1961) . ww= 7 . GOODFORD, P .J . and S . HSRMANSSN, J . .Phyaiol . (Londaa) 1~8, 416 w . (1961) .

8 . Q. .BIIRSSTOCS, D .J . DSfiHURST and S .S . SII~tON, J . Physiol . (Loadoa), ~6~, 210 (1963) . 9 . E .S . DANIEL, Caned . J . Biochem . phyaiol .~~, 206$ (1963) . 10 . W. McD . ARMSTRONG, Am . J . Phyaiol . 206, 470 (1964) . w=w

11 . D .F . BOHR, Pharmacol . Rev . 16, 85 (1964) . ww 12 . F . HAGEMEIJER, Arch . Iateraat . Phyaiol . Bioahim . ~~, 710 (1969) 13 " S. HAGEMEIJER, G .L . RORIYE and E . SCHOFFSNISLB, Arah . Iateraht . Phyaiol . Biochim . 7~, 453 (1965) " 14 . M .F . VILLA~MIL, Y . RSTTORI, L . BARAJAS aad C .R . =Lt!lIAN, Am . J . phyeiol . ~~4, 1104 (1968) .