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Rapid efflux of some quaternary ammonium compounds from cerebrospinal fluid
Life Sciences No, 10, pp . 515-521 * 1962 " Pergamon Press, Inc . Printed in the United States .
RAPID EFFLUX OF SOME QUATERNARY A1rIIr10NIUM COMPOUNDS FROM CEREBROSPINAL FLUID Lewis S . Schanker, Leon D . Prockop*, Jens Schouw* and Poonamchand Sisodia *** National Heart Institute, Bethesda, Maryland (Received 10 September 1962) RECENT studies of the fate of substances injected into the cerebrospina~. fluid (CSF) have indicated that solutes can pass from the fluid into the bloodstream in one or more of several ways . For
example, if a compound is lipid-soluble, it can diffuse across the lipid-like blood-CSF barrier+ - the same barrier that hinders the passage of many drugs into CSF after intravenous injection. 1 In addition s all solutes, regardless of their lipid-solubility, can
leave the CSF by flowing through the channels of the arachnoid villi as CSF drains through these structures to reach the ducal blood sinuses ; small molecules and ones as large as inulin or dextran all appear to leave the CSF at the same rate insofar as this route of exit is concerned . 2-6 A third way in which solutes may leave the CSF is by .specialized transport . For example, two highly ionized organic acids + phenol red and iodopyraeet (Diodrast), have been shown to pass from CSF to blood much more rapidly than do many other lipid-insoluble substances ; 3+7 moreover, the transport of the acids is depressed by ,p-aminohippurate, and the transport process is saturable . 7 Tho present report concerns the exit from CSF of three quaternary ammonium compounds : N1 -methylnicotinamide, hexamethonium, and decamethonium . The results suggest that these catioûs, like the above-mentioned anions, leave the CSF in part by a specialized transport process .
*Present address : Neurological Tnstitute, Columbia-Presbyterian Medical Center, New York . **Present address : Institute of Pharmacology, University of Copenhagen+ Denmark. ***Participant + U .S . Agency for International Development . Present address : Dept, of Pharmacology, Osmania Medical College, Hyderabad + India . 515
516
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
No . 10
Methods Male albino rabbits weighing 2 .1-2 .4 kg were anesthetized with ether and maintained under light anesthesia during the oa~peri-
ments . With the animal in the prone position and the head held in a clamp, the dura mater was exposed through a burr hole in the skull, and 10-30 ~1 of a solution of a radioactive-labeled substance (0 .2-2 .0 ~c) or N1-methylnicotinamide (NMN) injected into a lateral cerebral ventricle . Injections were made through a 27-guage needle * fused to a 10 or 50 ~tl syringe (Hamilton Co .), inserted with the
aid of a stereotaxic instrument . The doses of the various substances (mg/kg) were as follows : NMN chloride, 5 ; inulin-methoxyl-H3 , 0 .05 ; hexamethonium-methyl-C 14 diiodide, 0 .2 ; and decamethonium-methylC14 dibromide~ 0 .1 . At 1, 2 and 4 hr after the injection, O .l ml of CSF was withdrawn from the cisterna magna with a 22-guage needle inserted near the cranial vertex of the foramen magaum ; fluid in the needle (40 ~1) was removed just prior to drawing each sample . Both the cisternal and ventricular needles were held firmly in place throughout the 6 hr experiment to prevent leakage o~ CSF through the punctures . At the end of the experiment, 1 ml of CSF was withdrawn from the cisterna magna, and a small amount of the fluid also withdrawn through the injection needle to establish that the needle tip had remained within the ventricle . For the estimation of isotopically-labeled substances, 1 ml
of diluted CSF was added to 10 ml of a scintillation medium described by BrayB ~ anal the radioactivity measured with a Packard-Tri-Garb liquid scintillation spectrometer, model 314X . Samples containing both NMN and a radioactive substance could not be assayed in the above-mentioned medium because the NMN caused chemiluminescence resulting in spurious counts . 9 The difficulty was avoided by using a scintillation medium described by Gjone et a1 . 10 ; 0 .2 ml of diluted CSF was added to 18 ml of the medium, and the radioactivity measured as described above . For the estimation of Cl4 - and txitiumlabeled substances in the same sample, the simultaneous equation method of Okita ét al .ll was used with the instrument channels set at a balance point for C14 . In all measurements of radioactivity, the total number of counts exceeded 5000, and was at least 5 times the background . NMN was estimated fluorometrically according to the method of
Huff and Perlzweigl2 .
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
717
Results Disappearanç e of substances from CSF When NDiN and tritium-labeled inulin were injected together into a lateral cerebral ventricle of rabbits, the quaternary ammonium compound left tho CSF much more rapidly than did the saccharide (Fig . 1) . For example, after 2 hr, 9 per cent of the dose of NMN compared to 39 per cent of the inulin remained in 1 ml of cisternal
CSF ; and after 6 hr, only 0 .6 per cent of the NMN contrasted to 12 per cent of the inulin remained in 1 ml of the fluid . 100 80 60 40 N
c~
20
z W N O 0 W O F z W V
a I .0 W
o.lô
I I
I 2
TIME
I _- ._1._ 3 4 IN
HOURS
L___-L 5 6
FIG . 1 Disappearance of substances from CSF after intravenicular injection . A solution containing a quaternary ammonium compound and inulin was injected into a lateral ventricle of rabbits . At various times, samples of ciaternal CSF were trithdratvn and assayed for the two substances . Each 6 hr value is the mean of values obtained in 7-12 animals Each of the other points is the mean of values in 4-8 animals . Vertical brackets indicate the standard error of tha mAan .
51$
RAPID ï+;FFLUX OF SOME QUATERNARY AMMONIUM COMPOUND; fo . 10
T
Depressant effect of NDiN on efflux of methonium compounds from CSF Since the rate at which inulin leaves the CSF is a measure of the rate at zrizich solutes pass from CSF to blood by passive flow through the arachnoid villi, 6 the above result s. suggested that the three quaternary ammonium compounds had left the CSF in part by an additional process . To i~zvestigate the nature of the auxiliary mode of exit, the efflux of 1;üe methonium compounds was measured in the presence of
Effect of Ni~:h' on disappearance of hexamethonium from CSF . A solution containing NMAT, hexamethonium and inulin was injected into a lateral ventricle of rabbits . At various timcs~ samples of cisternal CSF were withdrawn and assayed for ïiexar~ethonium and inulin. Each point represents the mean of values obtained in 6-8 animals . Vertical brackets indicate the standard error of the mean . The curves for inulin and hexamethonium in the absence of NMN were taken from Fig . 1 .
No . 10
:3APID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
519
a high concentration of NMN. In a typical experiment, NMN (10 mg/ kg), tritiated inulin (0 .05 mg/kg), and either hexamethonium-C 14 (0 .2 mg/kg) or decamethonium-C14 (0 .1 mg/kg) were injected into a lateral cerebral ventricle ; samples of cisternal CSF were withdrawn at various times, and the radioactivity was measured . The results (Figs . 2 and 3) show that NMN depressed markedly the rates
of exit of hexamethonium and decamethonium . For example, the proportion of hexamethonium remaining in the CSF after 6 hr was increased almost fivefold in the presence of NMN ; and the proportion of decamethonium was increased about twofold . In contrast, tFie exit rate of inulin was not affected by the high concentration of
NMN .
c~
O 2 W N O C W O
H 2 W U W a
FIG . 3
Effect of NMN on disappearance of decamethonium from CSF . A solution containing NMN, decamethonium, and inulin was injected into a lateral ventricle of rabbits . At various times, samples of cisternal CSF were withdrawn and sasayed for decamethonium and inulin . Each point represents the mean of values obtained in 5 or 6 animals . Vertical brackets indicate the standard error of the mean . The curves for inulin and decamethonium in the absence of NMN were taken from Fig. l .
520
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
No, 10
Discussion Results of earlier studies, showing that lipid-insoluble substances with molecular weights ranging from 180 to 77,000 all escape from the CSF at the same rate by a process of filtration, 2-6 leave little doubt that NMN, hexamethonium, and decamethonium also leave by filtration at this rate . Accordingly, the observation that the quaternary ammonium compounds leave the CSF faster than inulin suggests that their efflux is the result of something more than simple filtration . Considering auxiliary modes of exit for the compounds
it seems permissible to rule out ready diffusion across
this boundary from blood to CSF, at extraordinarily slow rates .l3 The likelihood thus arises that the quaternary ammonium compounds are leaving the CSF in part by a specialized transport process . Moreover direct evidence for the existence of such a process is seen in the ability of NMN to depress markedly the rates of exit of inulin. Although the present study gives no indication of the locus
of the proposed transport mechanism, recent work showing that the epithelium of the choroid plexus actively transports phenol red and chlorphenol red into the capillary lumen in vitro l4 suggests
that it might be profitable to investigate this tissue as a possible site of transport for organic cations . The present results, together with those of earlier studies, l3 suggest that it is almost impossible for anions such as phenol red and Diodrast~ and cationa such as NMN, hexamethonium and deca methonium~ to attain a high concentration in the CSF after intravenous administration : in the first place, the diffusion of these
lipid-insoluble ions across the blood-CSF barrier is Beverly restricted because of the lipoid character of the barrier ; secondly, the small amounts of these substances that do reach the CSF are continually swept away by drainings of the fluid across the arachnoid villi ; and finally, these compounds appear to be readily expelled from the CSF by specialized transport processes . It is interesting to note the similarity between the central nervous system and the kidney with regard to the transport of organic compounds . Both organs have specialized mechanisms for transportinE anions, such as phenol red and Diodrast, and cations~ such as ND1N and hexamethonium .
No . 10
RAPID EFFLUX OP S©ME QUATERNARY AMMONIUM COMPOUNDS
521
Summary The quaternary ammonium compounds, Nl -methylnicotinamide (NMN), heaamethonium, sad decamethoaium, injected together with inulin into a lateral cerebral ventricle of rabbits, all leave the cerebrospinal fluid (CSF) more rapidly than inulin, and there-
fore more rapidly than cnn be accounted for by passive filtration alone . The ability of NMN to depress markedly the rates of exit of hexamethonium and decamethonium, ~rithout affecting the rate of exit of inulin, suggests that in addition to filtering across the arachnoid villa, the quaternary ammonium compounds leave by a specialized transport process .
Referencea 1 . S .E . MAYER, R.P . MAICKEL and B.B. BRODLE, J . Pharmacol . 12$ , 41 (1960) . 2 . L .D , . PROCKOP, L .S . SCHANKER and B .B . BRODLE, Sc_ i~-e . 1424 (1961) . 3 . L .D . PROC$OP, L .S . SCHANKER and B .B. BRODLE, J . Pharmacol . 1~, 266 (1962) . 4 . A .R. ROTFII~IAN, E .J . FREIREICH, J .R . GASKINS, C .S . PATLAK and D .P. BALL, Amer . J . Physiol . ®, 1145 (1961) . 5 . A,R. ROTHMAN, J .R. GASKINS, E .J . FREIREICH and D .P . BALL,
Na ~.~.: ~~ " ~+ 3a3 (1962) . 6 . L .D . PROCKOP sad L .S . SCHANKER, Life Sciences No . 4, 141 (1962) . 7 . J .R. PAPPENHEIMER, S .R. HEISEY and E .F . JORDAN, Amer . J . Ph`ol . ~, 1 (1961) . 8 . G .H. BRAY, Analyt . Biochem . 1, 279 (1960) . 9 . M. YAUGHN, D . 5TEINBERG and J . LOGAN, Science 126 , 446 (1960) . 10 . E . GJONE, H.G . VANCE and D .A . TURNER, Int . J . Appl . Rad . & Isotopes 8, 95 (1960) " G.T. ORITA, J .J . KABARA, F . RICHARDSON and G .Y . LEROY, Nucleonics ~, 111 0195?) . 12 . J .W. HUFF and W .A . PERLZWEIG, J . Hiol . Chem . ~, 395 (1943) . 13 . L .S . SCHANKER, Ann . Rev . Pharmacol . l, 29 (1961) . 14 . D .P . RALL and W . SHELDON, Biochem . Pharmacol . 11, 169 (1961) .
RAPID EFFLUX OF SOME QUATERNARY A1rIIr10NIUM COMPOUNDS FROM CEREBROSPINAL FLUID Lewis S . Schanker, Leon D . Prockop*, Jens Schouw* and Poonamchand Sisodia *** National Heart Institute, Bethesda, Maryland (Received 10 September 1962) RECENT studies of the fate of substances injected into the cerebrospina~. fluid (CSF) have indicated that solutes can pass from the fluid into the bloodstream in one or more of several ways . For
example, if a compound is lipid-soluble, it can diffuse across the lipid-like blood-CSF barrier+ - the same barrier that hinders the passage of many drugs into CSF after intravenous injection. 1 In addition s all solutes, regardless of their lipid-solubility, can
leave the CSF by flowing through the channels of the arachnoid villi as CSF drains through these structures to reach the ducal blood sinuses ; small molecules and ones as large as inulin or dextran all appear to leave the CSF at the same rate insofar as this route of exit is concerned . 2-6 A third way in which solutes may leave the CSF is by .specialized transport . For example, two highly ionized organic acids + phenol red and iodopyraeet (Diodrast), have been shown to pass from CSF to blood much more rapidly than do many other lipid-insoluble substances ; 3+7 moreover, the transport of the acids is depressed by ,p-aminohippurate, and the transport process is saturable . 7 Tho present report concerns the exit from CSF of three quaternary ammonium compounds : N1 -methylnicotinamide, hexamethonium, and decamethonium . The results suggest that these catioûs, like the above-mentioned anions, leave the CSF in part by a specialized transport process .
*Present address : Neurological Tnstitute, Columbia-Presbyterian Medical Center, New York . **Present address : Institute of Pharmacology, University of Copenhagen+ Denmark. ***Participant + U .S . Agency for International Development . Present address : Dept, of Pharmacology, Osmania Medical College, Hyderabad + India . 515
516
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
No . 10
Methods Male albino rabbits weighing 2 .1-2 .4 kg were anesthetized with ether and maintained under light anesthesia during the oa~peri-
ments . With the animal in the prone position and the head held in a clamp, the dura mater was exposed through a burr hole in the skull, and 10-30 ~1 of a solution of a radioactive-labeled substance (0 .2-2 .0 ~c) or N1-methylnicotinamide (NMN) injected into a lateral cerebral ventricle . Injections were made through a 27-guage needle * fused to a 10 or 50 ~tl syringe (Hamilton Co .), inserted with the
aid of a stereotaxic instrument . The doses of the various substances (mg/kg) were as follows : NMN chloride, 5 ; inulin-methoxyl-H3 , 0 .05 ; hexamethonium-methyl-C 14 diiodide, 0 .2 ; and decamethonium-methylC14 dibromide~ 0 .1 . At 1, 2 and 4 hr after the injection, O .l ml of CSF was withdrawn from the cisterna magna with a 22-guage needle inserted near the cranial vertex of the foramen magaum ; fluid in the needle (40 ~1) was removed just prior to drawing each sample . Both the cisternal and ventricular needles were held firmly in place throughout the 6 hr experiment to prevent leakage o~ CSF through the punctures . At the end of the experiment, 1 ml of CSF was withdrawn from the cisterna magna, and a small amount of the fluid also withdrawn through the injection needle to establish that the needle tip had remained within the ventricle . For the estimation of isotopically-labeled substances, 1 ml
of diluted CSF was added to 10 ml of a scintillation medium described by BrayB ~ anal the radioactivity measured with a Packard-Tri-Garb liquid scintillation spectrometer, model 314X . Samples containing both NMN and a radioactive substance could not be assayed in the above-mentioned medium because the NMN caused chemiluminescence resulting in spurious counts . 9 The difficulty was avoided by using a scintillation medium described by Gjone et a1 . 10 ; 0 .2 ml of diluted CSF was added to 18 ml of the medium, and the radioactivity measured as described above . For the estimation of Cl4 - and txitiumlabeled substances in the same sample, the simultaneous equation method of Okita ét al .ll was used with the instrument channels set at a balance point for C14 . In all measurements of radioactivity, the total number of counts exceeded 5000, and was at least 5 times the background . NMN was estimated fluorometrically according to the method of
Huff and Perlzweigl2 .
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
717
Results Disappearanç e of substances from CSF When NDiN and tritium-labeled inulin were injected together into a lateral cerebral ventricle of rabbits, the quaternary ammonium compound left tho CSF much more rapidly than did the saccharide (Fig . 1) . For example, after 2 hr, 9 per cent of the dose of NMN compared to 39 per cent of the inulin remained in 1 ml of cisternal
CSF ; and after 6 hr, only 0 .6 per cent of the NMN contrasted to 12 per cent of the inulin remained in 1 ml of the fluid . 100 80 60 40 N
c~
20
z W N O 0 W O F z W V
a I .0 W
o.lô
I I
I 2
TIME
I _- ._1._ 3 4 IN
HOURS
L___-L 5 6
FIG . 1 Disappearance of substances from CSF after intravenicular injection . A solution containing a quaternary ammonium compound and inulin was injected into a lateral ventricle of rabbits . At various times, samples of ciaternal CSF were trithdratvn and assayed for the two substances . Each 6 hr value is the mean of values obtained in 7-12 animals Each of the other points is the mean of values in 4-8 animals . Vertical brackets indicate the standard error of tha mAan .
51$
RAPID ï+;FFLUX OF SOME QUATERNARY AMMONIUM COMPOUND; fo . 10
T
Depressant effect of NDiN on efflux of methonium compounds from CSF Since the rate at which inulin leaves the CSF is a measure of the rate at zrizich solutes pass from CSF to blood by passive flow through the arachnoid villi, 6 the above result s. suggested that the three quaternary ammonium compounds had left the CSF in part by an additional process . To i~zvestigate the nature of the auxiliary mode of exit, the efflux of 1;üe methonium compounds was measured in the presence of
Effect of Ni~:h' on disappearance of hexamethonium from CSF . A solution containing NMAT, hexamethonium and inulin was injected into a lateral ventricle of rabbits . At various timcs~ samples of cisternal CSF were withdrawn and assayed for ïiexar~ethonium and inulin. Each point represents the mean of values obtained in 6-8 animals . Vertical brackets indicate the standard error of the mean . The curves for inulin and hexamethonium in the absence of NMN were taken from Fig . 1 .
No . 10
:3APID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
519
a high concentration of NMN. In a typical experiment, NMN (10 mg/ kg), tritiated inulin (0 .05 mg/kg), and either hexamethonium-C 14 (0 .2 mg/kg) or decamethonium-C14 (0 .1 mg/kg) were injected into a lateral cerebral ventricle ; samples of cisternal CSF were withdrawn at various times, and the radioactivity was measured . The results (Figs . 2 and 3) show that NMN depressed markedly the rates
of exit of hexamethonium and decamethonium . For example, the proportion of hexamethonium remaining in the CSF after 6 hr was increased almost fivefold in the presence of NMN ; and the proportion of decamethonium was increased about twofold . In contrast, tFie exit rate of inulin was not affected by the high concentration of
NMN .
c~
O 2 W N O C W O
H 2 W U W a
FIG . 3
Effect of NMN on disappearance of decamethonium from CSF . A solution containing NMN, decamethonium, and inulin was injected into a lateral ventricle of rabbits . At various times, samples of cisternal CSF were withdrawn and sasayed for decamethonium and inulin . Each point represents the mean of values obtained in 5 or 6 animals . Vertical brackets indicate the standard error of the mean . The curves for inulin and decamethonium in the absence of NMN were taken from Fig. l .
520
RAPID EFFLUX OF SOME QUATERNARY AMMONIUM COMPOUNDS
No, 10
Discussion Results of earlier studies, showing that lipid-insoluble substances with molecular weights ranging from 180 to 77,000 all escape from the CSF at the same rate by a process of filtration, 2-6 leave little doubt that NMN, hexamethonium, and decamethonium also leave by filtration at this rate . Accordingly, the observation that the quaternary ammonium compounds leave the CSF faster than inulin suggests that their efflux is the result of something more than simple filtration . Considering auxiliary modes of exit for the compounds
it seems permissible to rule out ready diffusion across
this boundary from blood to CSF, at extraordinarily slow rates .l3 The likelihood thus arises that the quaternary ammonium compounds are leaving the CSF in part by a specialized transport process . Moreover direct evidence for the existence of such a process is seen in the ability of NMN to depress markedly the rates of exit of inulin. Although the present study gives no indication of the locus
of the proposed transport mechanism, recent work showing that the epithelium of the choroid plexus actively transports phenol red and chlorphenol red into the capillary lumen in vitro l4 suggests
that it might be profitable to investigate this tissue as a possible site of transport for organic cations . The present results, together with those of earlier studies, l3 suggest that it is almost impossible for anions such as phenol red and Diodrast~ and cationa such as NMN, hexamethonium and deca methonium~ to attain a high concentration in the CSF after intravenous administration : in the first place, the diffusion of these
lipid-insoluble ions across the blood-CSF barrier is Beverly restricted because of the lipoid character of the barrier ; secondly, the small amounts of these substances that do reach the CSF are continually swept away by drainings of the fluid across the arachnoid villi ; and finally, these compounds appear to be readily expelled from the CSF by specialized transport processes . It is interesting to note the similarity between the central nervous system and the kidney with regard to the transport of organic compounds . Both organs have specialized mechanisms for transportinE anions, such as phenol red and Diodrast, and cations~ such as ND1N and hexamethonium .
No . 10
RAPID EFFLUX OP S©ME QUATERNARY AMMONIUM COMPOUNDS
521
Summary The quaternary ammonium compounds, Nl -methylnicotinamide (NMN), heaamethonium, sad decamethoaium, injected together with inulin into a lateral cerebral ventricle of rabbits, all leave the cerebrospinal fluid (CSF) more rapidly than inulin, and there-
fore more rapidly than cnn be accounted for by passive filtration alone . The ability of NMN to depress markedly the rates of exit of hexamethonium and decamethonium, ~rithout affecting the rate of exit of inulin, suggests that in addition to filtering across the arachnoid villa, the quaternary ammonium compounds leave by a specialized transport process .
Referencea 1 . S .E . MAYER, R.P . MAICKEL and B.B. BRODLE, J . Pharmacol . 12$ , 41 (1960) . 2 . L .D , . PROCKOP, L .S . SCHANKER and B .B . BRODLE, Sc_ i~-e . 1424 (1961) . 3 . L .D . PROC$OP, L .S . SCHANKER and B .B. BRODLE, J . Pharmacol . 1~, 266 (1962) . 4 . A .R. ROTFII~IAN, E .J . FREIREICH, J .R . GASKINS, C .S . PATLAK and D .P. BALL, Amer . J . Physiol . ®, 1145 (1961) . 5 . A,R. ROTHMAN, J .R. GASKINS, E .J . FREIREICH and D .P . BALL,
Na ~.~.: ~~ " ~+ 3a3 (1962) . 6 . L .D . PROCKOP sad L .S . SCHANKER, Life Sciences No . 4, 141 (1962) . 7 . J .R. PAPPENHEIMER, S .R. HEISEY and E .F . JORDAN, Amer . J . Ph`ol . ~, 1 (1961) . 8 . G .H. BRAY, Analyt . Biochem . 1, 279 (1960) . 9 . M. YAUGHN, D . 5TEINBERG and J . LOGAN, Science 126 , 446 (1960) . 10 . E . GJONE, H.G . VANCE and D .A . TURNER, Int . J . Appl . Rad . & Isotopes 8, 95 (1960) " G.T. ORITA, J .J . KABARA, F . RICHARDSON and G .Y . LEROY, Nucleonics ~, 111 0195?) . 12 . J .W. HUFF and W .A . PERLZWEIG, J . Hiol . Chem . ~, 395 (1943) . 13 . L .S . SCHANKER, Ann . Rev . Pharmacol . l, 29 (1961) . 14 . D .P . RALL and W . SHELDON, Biochem . Pharmacol . 11, 169 (1961) .