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Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume
Life Sciences, Vol. 37, pp. 561-566 Printed in the U.S.A.
Pergamon Press
INCREASED DIGITALIS-LIKE ACTIVITY IN HUMAN CEREBROSPINAL FLUID AFTER EXPANSION OF THE EXTRACELLULAR FLUID VOLUME Jose A. Halperin*, Ana M. Martin & Salavador Malave Unidad de Investigaciones and Unidad de Radiodiagnostico, Centro Medico Docente La Trinidad, A.P. 80474, Caracas 1080 A, Venezuela (Received in final form June 4, 1985) Summary The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described by our laboratdry. Human CSF samples, drawn before and 30 minutes after tile intravenous infusion of i liter of either saline or glucose solutions, were assayed for digitalislike activity by inhibition of either the 86Rb+ uptake into human erythrocytes or by the activity of a purified Na+ - K + ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading. Data originally reported by our laboratory showed that an endogenous inhibitor of the Na + - K + ATPase is present in human cerebrospinal fluid (CSF) (i). /Briefly, human CSF was found: i) to reduce both active Na + efflux and 86Rb + influx in human red cells and ii) to inhibit the in vitro activity of a purified Na + - K + ATPase (i). These observations have recently been confirmed by Lichtstein et al. (2) ~lo additionally have shown that human CSF also inhibits the binding of [3H] ouabain to rat brain synaptosomes. Humoral inhibitors of active Na + transport have been extracted from different tissues (3) and from plasma (4) and urine (5) of volume expanded subjects and experimental animals (6,7) and have been proposed as playing a role in the pathogenesis of some forms of Na + dependent hypertension (8,9). We report here that the acute expansion of the extracellular fluid volume with Na + increases the Na + - K+ ATPase inhibitory activity of human CSF. Methods CSF was obtained after overnight fasting from normotensive male and female donors to whom a lumber myelography was indicated as a diagnostic procedure for possible spinal root compression. Informed consent was obtained in all cases.
* Present Address: Dept. of Physiology, Harvard Medical School, 25 Shattuck Street, Boston, lJ~ 02115 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
562
Increased Digitalis-like Activity in Human CSF
Vol. 37, No. 6, 1985
At the moment of the lumbar puncture a preexpansion sample was drawn. Immediately thereafter, 1 liter of either isotonic saline solution (NaCI 150 mM) or 5% glucose solution was infused intravenously at a rate of 65 ml/min. Samples were then frozen at -70 C or immediately assayed for ouabain-like activity by the inhibition of either the 86Rb+ uptake into human red cells or the purified Na + - K + ATPase. The ionic composition of each CSF sample was determined and the final concentration of Na + and K+ was adjusted to a value similar to the control medium of the respective experiment by adding the required amount of 1 M NaCI or KCI solution. Rubidium-86 uptake into human red cells: The 86Rb + uptake into human red cells was estimated by the following procedure: triplicate aliquots of washed packed red blood cells were resuspended at a final hematocrit of 10% in 200~i of either a) control medium (NaCI 150 mM, KC1 i0 mM, MgCI 2 1 mM, glucose 10mM, Tris-HCl i0 mM, pH 7.4 at 37 C) or b) CSF (with NaCI, KCI and Tris-HCl concentrations adjusted to those of the control medium by adding to 1 ml of CSF 4~i, 7~i, and i0~i of the respective 1 M solution). Samples were then incubated at 37 C for 1 hour with 105 cpm of 6Rb (New England Nuclear, specific activity 1.09 mCi/mg). The influx of 86Rb was terminated by washing the erythrocytes three times with ice-cold isotonic choline chloride and the incorporated radioactivity was eluted and counted as previously described (i). Rubidium uptake in the control medium was taken as 100% and the results expressed as the percentage of inhibition of the 86Rb + uptake: Rb + uptake in CSF i00
-
[ .............................
]
x
i00
Rb + uptake in control medium Na + - K+ ATPase inhibition: The activity of the purified Na + - K + ATPase (ATP phosphoydrolase E.C. 3613, grade IV, Sigma Co.) was estimated by the rate of [~-32p] - ATP hydrolysis after incubating the enzyme for 20 minutes at 37 C in either control medium or CSF with the ionic and buffer concentrations adJusted to those of the control medium by addition of the required amount of a 1 M solution. In both assay media the final reaction conditions were as follows: NaCI 150 n~nol, KCI 20 mmol, MgCI 2 5.2 mmol, Ca ++ 0.6 mmol, EGTA 1.5 mmol, Tris-HCl 50 mmol, pH 7.4 at 37 C. The protocol for measuring the Na + - K + ATPase was previously described (i). The enzymatic activity was estimated by the ratio (x i00) between the 32_ Phosphorus liberated in the supernatant fluid and the total radioactivity added to each sample. Results were expressed as the percentage of inhibition of the Na + - K+ ATPase activity. Activity in CSF i00
-
[ .............................
]
x
i00
Activity in control medium Results The ionic composition of the CSF samples did not change significantly after the infusion of 1 liter of either saline or glucose solution (Table i).
Vol. 37, No. 6, 1985
Increased Digltalis-like Activity in Human CSF
563
TABLE 1 Concentration (mM) lon
Pre
Post saline (n=14) 147 ± 2 2.8 ± .i i ± .01
(n=26) 146 + 2 2.8 ± .05 i ± .01
Na + K+ Ca + +
glucose (n=13) 147 + 2 2.8 + .i i ± .01
Values are means ± sem All the CSF samples drawn before expansion of the ECFV, inhibited the 86Rb+ uptake and the Na + - K+ ATPase activity with respect to the corresponding control medium, in accordance with our previous observation (i). The CSF inhibitory effect on the Rh + (K+) influx and on the enzymatic activity significantly increased in the postexpansion samples drawn after the infusion of saline solutions while it did not change in the CSF samples drawn after the infusion of isotonic glucose (Figures i & 2). Figures i and 2 also show that the relationship between the inhibitory activity of the POSTexpansion and PREexpansion CSF samples (POST/PRE ratio) was in both assays, around 30% higher in sodium-infused subjects than in glucoseinfused ones.
pre
iQo.
60 1
I
post
I
n=7
pre
]
I
post
n=7
'~
÷
~
-4-
4o
T
OJ
'g.m
I
1.5'i ~
GLIICOSE
.~ALIN[
FIG. 1 Increased inhibition of the 86Rb+ uptake into erythrocytes by human CSF after expansion of the ECFV. The o6 Rb + uptake by human red cells was measured in PRE ( o ; e ) and POST (A;A) expansion samples, drawn before and 30 minutes after the infusion of either glucose ( o _ A ) or saline ( e _ ; ) solution. The uptake in control medium was taken as 100% and the percent inhibition produced by CSF was calculated as described in methods. Horizontal bars represent means ± sem. * p < 0.001 as evaluated by Student's t test for paired data.
564
Increased Digitalis-like
IoQ ] 7D
~-
I
Activity
in Human CSF
pre
post
I
I
]
Vol.
37, No.
pre
post
I
!
6, 1985
n =7
611
N 3n
+er ~
nJ 1.5'
GLIICEISE
. ALINE
FIG 2 Increased Na + - K + ATPase inhibition
by human CSF after expansion of the
ECFV. The activity of the enzyme was estimated by the hydrolysis of [~_32p]_ ATP in control or in CSF samples drawn prior to (PRE o;e) and 30 minutes after (POST A:A) the infusion of glucose ( o _ A ) or saline ( e _ A ) solution. The enzymatic activity in control medium was taken as 100% and the percent inhibition produced by CSF was calculated as described in methods. Horizontal bars represent means ± sem. p < 0.001 as evaluated by Student's t test for paired data. Both pre and post expansion CSF samples inhibited the ouabain sensitive component of the 86Rb+ uptake (data not shown); comparison of this effect to the ATPase inhibition produced by each specimen showed a positive linear correlation (r=0.75;p<0.001). Discussion An earlier report from our laboratory showed the existence of a Na + - K + ATPase inhibitor in human CSF. The present study shows that this inhibitory activity increases after the infusion of saline solution but remains unchanged when the same amount of glucose was injected. These results indicate that the inhibitory agent present in human CSF has an endogenous source and that it could be related to volume and sodium regulation. Because sodium ions are almost confined to the extracellular fluid and because glucose penetrates the intracellular space, the infusion of isotonic saline is likely to induce a larger expansion of the extracellular fluid volume than the infusion of the same amount of isotonic glucose. However, a specific response to sodium ions might also be involved in the increased digitalis-like activity observed after sodium loading in human CSF. The physiological responses to acute sodium loading include natriuresis and hemodynamic changes like increased cardiac output, stroke volume and peripheral resistance (i0); these responses are similar to some pharmac~logic
Vol. 37, No. 6, 1985
Increased Digitalis-like
Activity
in Human CSF
565
effects of the digitalis compounds such as natriuresis (ii)~ the positive inotropic action on cardiac muscle and the constrictive effect on blood vessels (12). Inhibition of the Na + pump would induce the latter effect either by reducing the Ca ++ efflux through the Na + - Ca++ exchange as proposed by Blaustein (13), or by electrogenic depolarization (14) and increased noradrenergic activity (15). Norepinephrine release and uptake by the adrenergic synaptosomes are Na + and K + dependent mechanisms, both influenced by ouabain (16,17). Plasma extracts derived from different Na + - loaded animals exhibit digitalis-like properties in different experimental assays. These extracts reduce: i) the short circuit currents of the toad urinary bladder (18), ii) the in vitro activity of a purified Na + - K+ ATPase (19) and iii) the 86Rb+ uptake by the rat tail artery (8). These plasma extracts also displace the digoxin binding to a specific antidigoxin antibody (6). Thus, an endogenous inhibitor of the Na + pump could be one possible link between a high Na + intake and some of the above mentioned responses to a sodium load. The plasma concentration of the digitalis-like compound appears to be influenced by the periventricular anteroventral third ventricle (AV3V) area of the brain (20), which has also been shown to be important in the development of certain forms of experimental hypertension (21). Experimental animals with AV3V lesions fail to exhibit natriuresis and plasmatic digitalis-like activity after expansion of the extracellular fluid volume with sodium. Interestingly, the drinking behaviour elicited by acute Na + loading (22,23) also requires the integrity of the hypothalamic periventricular region (24). Moreover, in the rat, the intraventricular injection of angiotensin II, which induces an immediate drinking response (24) has recently been found to raise the digitalislike activity present in the plasma of these animals (V. Nahmod, personal communication). The recent identification in human and rat atrial tissues of natriuretic and muscle-relaxing peptides (25) lacking Na ÷ - K + ATPase inhibitory properties (26), together with the growing evidence indicating the existence of an endogenous Na+ - K + ATPase inhibitor released or activated by ECFV expansion, suggests that at least two different agents might be involved in the physiological system controlling sodium excretion and volume homeostasis. Our finding of an increased digitalis-like activity in human CSF after acute sodium loading, together with the presence of atriopeptin im~unoreactive neurons in the AV3V region of the brain (27) further supports this hypothesis. The earlier report of a circulating inhibitor of the sodium pump (28) and other ouabain-insensitive sodium transport abnormalities (30.31) - in hypertensive subjects increases the interest of the present communication. Acknowledgement The authors are grateful to Dr. M. Piras, R. Piras and P. Pulido for the continuous interest, critical discussion and review of the manuscript. We express our gratitude to Gloria Osorio for expert technical assistance. This study was partially supported by Research Grant SI-1552 from CONICIT (Venezuela) and by Centro Medico Docente La Trinidad, Caracas, Venezuela.
566
Increased Digitalis-like Activity
in Human CSF
Vol.
37, No. 6, 1985
References i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
J.A. HALPERIN, R. SHAEFFER, L. GALVEZ and S. MALAVE, Proc. Natl. Acad. Sci. USA. 80 6101-6104 (1983). D. LICHTSTEIN, D. MINC, A, BOURRIT, J. DEUTSCH, S.J.D. KARLISH, H. BELMAKER R. RIMON and J. PALO, Brain Res. 325 13-19 (1985). G.T. HAUPERT and J.M. SANCHO, Proc. Natl. Acad. Sci. USA 76 4685-4660 (1979) V.M. BUCKALEW and K.A. GRUBER, Natriuretic hormone. In Epstein M., ed. The kidney in the liver disease. 2d. p. 479. Elsevier Science Publishing Co, New York (1983). D. KLINGMULER, E. WEILER, H.J. KRAMER. Kiln. Wochensch. 60 1249-1253 (1982). K.A. GRUBER, J.M. WHITAKER and V.M. BUCKALEW, Nature 287 743-745 (1980). V.M. BUCKALEW and D.B. NELSON, Kidney Int. 5 12-22 (1974). F.J. HADDY and M.B. PAMNANI, Fed. Proc. 42 2673-2680 (1983). H.E. de WARDENER and G.A. MACGREGOR, Kidney Int. 18 1-9 (1980). F.J. HADDY, Am. J. Med. 69 746-758 (1980). J.B. HOOK, Proc. Soc. Exp. Biol. Med. 131 731-734 (1969). F.J. HADDY, Hypertensive Mechanisms. Procedings of the 4th International Symposium on rats with spontaneous hypertension and related studies, p.217 F.K. Shattauer Verlag Stuttgart, New York (1982). M.P. BLAUSTEIN, Am. J. Physiol. 232 C165-C173 (1977). F.J. HADDY and H.W. OVERBECK, Life Sci. 19 935-948 (1976). F.J. HADDY, M.B. PAMNANI and D.L. CLOUGH, Clin. Exp. Hypertension 1 295336 (1978). V.K. SHARMA and S.P. BANERJEE, Eur. J. Pharmacol. 417-419 (1977). Y. NAKAZATO, A. ORGA and Y. ANODA, J. Physiol. 278 45-54 (1978). V.M. BUCKALEW, F.J. MARTINEZ and W.E. GREEN, J. Clin. Invest. 49 926-935 (1970). H.C. GONICK, H.J. KRAMER, E. PAUL and E. LU, Clin. Sci. Mol. Med. 53 329334 (1977). S.L. BEALER, J.R. HAYWOOD, K.A. GRUBER, V.M. BUCKALEW, G.D. FINK, M.J, BRODY and A.K. JOHNSON, Am. J. Physio. 244 RSI-R57 (1983). M.J. BRODY, G.D. FINK, J. BUGGY, J.R. HAYWOOD, F.J. GORDON and A.K. JOHNSON, Cir. Res. 43 112-113 (1978). D.J. GOLDSTEIN and J.A. HALPERIN, Nature 267, 250-252 (1977). J.T. FITZIMONDS, J. Physiol. London 155 563-566 (1961). M.I. PHILLIPS, W.E. HOFFMAN and S.L. BEALER, Fed. Proc. 41 2520-2527
(1982). 25. 26. 27. 28. 29. 30. 31.
M.G. CURRIE, D.M. GELLER, B.R. COLE, N.R. SIEGEL, K.F. FOX, S.P. ADAMS, S.R. EUBANKS, G.R. GALLUPI and P. NEEDLEMAN, Science 223 67-69 (1984). G. THIBAULT, R. GARCIA, M. CANTIN and J. GENEST, Hypertension 5 (suppl.l) 1-81 (1983). C.B. SAPER, D.G. STANDAERT, M.G. CURRIE, D. SCHWARTZ, D.M. GELLER and P. NEEDLEMAN, Science 227 1047-1049 (1985). J.M. HAMLYN, R. RINGEL, J. SHAEFFER, P.D. LEVINSON, B.P. HAMILTON, A. KOWARSKI and M.P. BLAUSTEIN, Nature 300 650-652 (1982). M. CANESSA, N. ADRAGNA, H.S. SOLOMON, T.M. CONOLLY, D.C. TOSTESON, N. Engl. J. Med. 302 772-772 (1980). R.P. GARAY, G. DAGHER, M.G. PERNOLLET, M.A. DEVYNK and P.H. MEYER, Nature 284 281-282 (1980). J.A. HALPERIN and L. GALVEZ, Acta Cient. Venez. 32 196 (1981).
Pergamon Press
INCREASED DIGITALIS-LIKE ACTIVITY IN HUMAN CEREBROSPINAL FLUID AFTER EXPANSION OF THE EXTRACELLULAR FLUID VOLUME Jose A. Halperin*, Ana M. Martin & Salavador Malave Unidad de Investigaciones and Unidad de Radiodiagnostico, Centro Medico Docente La Trinidad, A.P. 80474, Caracas 1080 A, Venezuela (Received in final form June 4, 1985) Summary The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described by our laboratdry. Human CSF samples, drawn before and 30 minutes after tile intravenous infusion of i liter of either saline or glucose solutions, were assayed for digitalislike activity by inhibition of either the 86Rb+ uptake into human erythrocytes or by the activity of a purified Na+ - K + ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading. Data originally reported by our laboratory showed that an endogenous inhibitor of the Na + - K + ATPase is present in human cerebrospinal fluid (CSF) (i). /Briefly, human CSF was found: i) to reduce both active Na + efflux and 86Rb + influx in human red cells and ii) to inhibit the in vitro activity of a purified Na + - K + ATPase (i). These observations have recently been confirmed by Lichtstein et al. (2) ~lo additionally have shown that human CSF also inhibits the binding of [3H] ouabain to rat brain synaptosomes. Humoral inhibitors of active Na + transport have been extracted from different tissues (3) and from plasma (4) and urine (5) of volume expanded subjects and experimental animals (6,7) and have been proposed as playing a role in the pathogenesis of some forms of Na + dependent hypertension (8,9). We report here that the acute expansion of the extracellular fluid volume with Na + increases the Na + - K+ ATPase inhibitory activity of human CSF. Methods CSF was obtained after overnight fasting from normotensive male and female donors to whom a lumber myelography was indicated as a diagnostic procedure for possible spinal root compression. Informed consent was obtained in all cases.
* Present Address: Dept. of Physiology, Harvard Medical School, 25 Shattuck Street, Boston, lJ~ 02115 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
562
Increased Digitalis-like Activity in Human CSF
Vol. 37, No. 6, 1985
At the moment of the lumbar puncture a preexpansion sample was drawn. Immediately thereafter, 1 liter of either isotonic saline solution (NaCI 150 mM) or 5% glucose solution was infused intravenously at a rate of 65 ml/min. Samples were then frozen at -70 C or immediately assayed for ouabain-like activity by the inhibition of either the 86Rb+ uptake into human red cells or the purified Na + - K + ATPase. The ionic composition of each CSF sample was determined and the final concentration of Na + and K+ was adjusted to a value similar to the control medium of the respective experiment by adding the required amount of 1 M NaCI or KCI solution. Rubidium-86 uptake into human red cells: The 86Rb + uptake into human red cells was estimated by the following procedure: triplicate aliquots of washed packed red blood cells were resuspended at a final hematocrit of 10% in 200~i of either a) control medium (NaCI 150 mM, KC1 i0 mM, MgCI 2 1 mM, glucose 10mM, Tris-HCl i0 mM, pH 7.4 at 37 C) or b) CSF (with NaCI, KCI and Tris-HCl concentrations adjusted to those of the control medium by adding to 1 ml of CSF 4~i, 7~i, and i0~i of the respective 1 M solution). Samples were then incubated at 37 C for 1 hour with 105 cpm of 6Rb (New England Nuclear, specific activity 1.09 mCi/mg). The influx of 86Rb was terminated by washing the erythrocytes three times with ice-cold isotonic choline chloride and the incorporated radioactivity was eluted and counted as previously described (i). Rubidium uptake in the control medium was taken as 100% and the results expressed as the percentage of inhibition of the 86Rb + uptake: Rb + uptake in CSF i00
-
[ .............................
]
x
i00
Rb + uptake in control medium Na + - K+ ATPase inhibition: The activity of the purified Na + - K + ATPase (ATP phosphoydrolase E.C. 3613, grade IV, Sigma Co.) was estimated by the rate of [~-32p] - ATP hydrolysis after incubating the enzyme for 20 minutes at 37 C in either control medium or CSF with the ionic and buffer concentrations adJusted to those of the control medium by addition of the required amount of a 1 M solution. In both assay media the final reaction conditions were as follows: NaCI 150 n~nol, KCI 20 mmol, MgCI 2 5.2 mmol, Ca ++ 0.6 mmol, EGTA 1.5 mmol, Tris-HCl 50 mmol, pH 7.4 at 37 C. The protocol for measuring the Na + - K + ATPase was previously described (i). The enzymatic activity was estimated by the ratio (x i00) between the 32_ Phosphorus liberated in the supernatant fluid and the total radioactivity added to each sample. Results were expressed as the percentage of inhibition of the Na + - K+ ATPase activity. Activity in CSF i00
-
[ .............................
]
x
i00
Activity in control medium Results The ionic composition of the CSF samples did not change significantly after the infusion of 1 liter of either saline or glucose solution (Table i).
Vol. 37, No. 6, 1985
Increased Digltalis-like Activity in Human CSF
563
TABLE 1 Concentration (mM) lon
Pre
Post saline (n=14) 147 ± 2 2.8 ± .i i ± .01
(n=26) 146 + 2 2.8 ± .05 i ± .01
Na + K+ Ca + +
glucose (n=13) 147 + 2 2.8 + .i i ± .01
Values are means ± sem All the CSF samples drawn before expansion of the ECFV, inhibited the 86Rb+ uptake and the Na + - K+ ATPase activity with respect to the corresponding control medium, in accordance with our previous observation (i). The CSF inhibitory effect on the Rh + (K+) influx and on the enzymatic activity significantly increased in the postexpansion samples drawn after the infusion of saline solutions while it did not change in the CSF samples drawn after the infusion of isotonic glucose (Figures i & 2). Figures i and 2 also show that the relationship between the inhibitory activity of the POSTexpansion and PREexpansion CSF samples (POST/PRE ratio) was in both assays, around 30% higher in sodium-infused subjects than in glucoseinfused ones.
pre
iQo.
60 1
I
post
I
n=7
pre
]
I
post
n=7
'~
÷
~
-4-
4o
T
OJ
'g.m
I
1.5'i ~
GLIICOSE
.~ALIN[
FIG. 1 Increased inhibition of the 86Rb+ uptake into erythrocytes by human CSF after expansion of the ECFV. The o6 Rb + uptake by human red cells was measured in PRE ( o ; e ) and POST (A;A) expansion samples, drawn before and 30 minutes after the infusion of either glucose ( o _ A ) or saline ( e _ ; ) solution. The uptake in control medium was taken as 100% and the percent inhibition produced by CSF was calculated as described in methods. Horizontal bars represent means ± sem. * p < 0.001 as evaluated by Student's t test for paired data.
564
Increased Digitalis-like
IoQ ] 7D
~-
I
Activity
in Human CSF
pre
post
I
I
]
Vol.
37, No.
pre
post
I
!
6, 1985
n =7
611
N 3n
+er ~
nJ 1.5'
GLIICEISE
. ALINE
FIG 2 Increased Na + - K + ATPase inhibition
by human CSF after expansion of the
ECFV. The activity of the enzyme was estimated by the hydrolysis of [~_32p]_ ATP in control or in CSF samples drawn prior to (PRE o;e) and 30 minutes after (POST A:A) the infusion of glucose ( o _ A ) or saline ( e _ A ) solution. The enzymatic activity in control medium was taken as 100% and the percent inhibition produced by CSF was calculated as described in methods. Horizontal bars represent means ± sem. p < 0.001 as evaluated by Student's t test for paired data. Both pre and post expansion CSF samples inhibited the ouabain sensitive component of the 86Rb+ uptake (data not shown); comparison of this effect to the ATPase inhibition produced by each specimen showed a positive linear correlation (r=0.75;p<0.001). Discussion An earlier report from our laboratory showed the existence of a Na + - K + ATPase inhibitor in human CSF. The present study shows that this inhibitory activity increases after the infusion of saline solution but remains unchanged when the same amount of glucose was injected. These results indicate that the inhibitory agent present in human CSF has an endogenous source and that it could be related to volume and sodium regulation. Because sodium ions are almost confined to the extracellular fluid and because glucose penetrates the intracellular space, the infusion of isotonic saline is likely to induce a larger expansion of the extracellular fluid volume than the infusion of the same amount of isotonic glucose. However, a specific response to sodium ions might also be involved in the increased digitalis-like activity observed after sodium loading in human CSF. The physiological responses to acute sodium loading include natriuresis and hemodynamic changes like increased cardiac output, stroke volume and peripheral resistance (i0); these responses are similar to some pharmac~logic
Vol. 37, No. 6, 1985
Increased Digitalis-like
Activity
in Human CSF
565
effects of the digitalis compounds such as natriuresis (ii)~ the positive inotropic action on cardiac muscle and the constrictive effect on blood vessels (12). Inhibition of the Na + pump would induce the latter effect either by reducing the Ca ++ efflux through the Na + - Ca++ exchange as proposed by Blaustein (13), or by electrogenic depolarization (14) and increased noradrenergic activity (15). Norepinephrine release and uptake by the adrenergic synaptosomes are Na + and K + dependent mechanisms, both influenced by ouabain (16,17). Plasma extracts derived from different Na + - loaded animals exhibit digitalis-like properties in different experimental assays. These extracts reduce: i) the short circuit currents of the toad urinary bladder (18), ii) the in vitro activity of a purified Na + - K+ ATPase (19) and iii) the 86Rb+ uptake by the rat tail artery (8). These plasma extracts also displace the digoxin binding to a specific antidigoxin antibody (6). Thus, an endogenous inhibitor of the Na + pump could be one possible link between a high Na + intake and some of the above mentioned responses to a sodium load. The plasma concentration of the digitalis-like compound appears to be influenced by the periventricular anteroventral third ventricle (AV3V) area of the brain (20), which has also been shown to be important in the development of certain forms of experimental hypertension (21). Experimental animals with AV3V lesions fail to exhibit natriuresis and plasmatic digitalis-like activity after expansion of the extracellular fluid volume with sodium. Interestingly, the drinking behaviour elicited by acute Na + loading (22,23) also requires the integrity of the hypothalamic periventricular region (24). Moreover, in the rat, the intraventricular injection of angiotensin II, which induces an immediate drinking response (24) has recently been found to raise the digitalislike activity present in the plasma of these animals (V. Nahmod, personal communication). The recent identification in human and rat atrial tissues of natriuretic and muscle-relaxing peptides (25) lacking Na ÷ - K + ATPase inhibitory properties (26), together with the growing evidence indicating the existence of an endogenous Na+ - K + ATPase inhibitor released or activated by ECFV expansion, suggests that at least two different agents might be involved in the physiological system controlling sodium excretion and volume homeostasis. Our finding of an increased digitalis-like activity in human CSF after acute sodium loading, together with the presence of atriopeptin im~unoreactive neurons in the AV3V region of the brain (27) further supports this hypothesis. The earlier report of a circulating inhibitor of the sodium pump (28) and other ouabain-insensitive sodium transport abnormalities (30.31) - in hypertensive subjects increases the interest of the present communication. Acknowledgement The authors are grateful to Dr. M. Piras, R. Piras and P. Pulido for the continuous interest, critical discussion and review of the manuscript. We express our gratitude to Gloria Osorio for expert technical assistance. This study was partially supported by Research Grant SI-1552 from CONICIT (Venezuela) and by Centro Medico Docente La Trinidad, Caracas, Venezuela.
566
Increased Digitalis-like Activity
in Human CSF
Vol.
37, No. 6, 1985
References i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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