Naunyn-Schmiedeberg'sArch. Pharmaeol. 290, 99-- 105 (1975) 9 by Springer-Verlag 1975
Short Communication Steady.State Concentrations of Choline and Acetylcholine in Rat Brain Parts during a Constant Rate Infusion of Deuterated Choline G. Racagni, M. Trabucchi, a n d D. L. Cheney Laboratory of Preclinical Pharmacology, National Institute of i~[ental Health, Saint Elizabeths Hospital, Washington, D.C. Received March 7 / Accepted June 11, 1975
Summary. An intravenous infusion of deuteratod choline at constant rate for 6 rain (5 or 25 ~zmoleskg -1 rain-1) significantly increases the concentration of choline in plasma,, occipital cortex and striatum. Both 5 and 25 ~moles kg -~ rain-1 increase the concentration of acetylcholine in cortex but only 25 [zmoles kg-1 min-1 increases the acetyleholine content in striatum. In contrast, 1 [zrnolekg -1 rain-1 does not change the choline or acetylcholine content in cortex or striatum. A single pulse injection of choline (200 tzmoles kg-1) causes a significant increase in the concentration of choline in striatum 30 see following injection. The choline content returns to normal values within 2 rain. These studies show that when a pulse injection of a non-tracer dose of radioactive choline is used to measure brain aeetylcholine turnover rate the maintenance of steady state must be verified within seconds after the pulse injection of radioactive choline. When constant infusion of deuterated choline is used to measure turnover rate of acetylcholine in the brain of rats, a dose of I ~Lmolekg-1 rain-1 appears to be a maximal infusion rate. Key words: Choline -- Acetylcholine -- Infusion -- Striatum -- Occipital Cortex -- Mass Fragmentography.
Introduction R e c e n t studies i n whole b r a i n of g u i n e a pigs ( H a u b r i c h et al., 1974) a n d mice ( J e n d e n et al., 1974) suggested t h a t the s t e a d y - s t a t e concent r a t i o n s of choline (Ch) a n d acetylcholine (ACh) did n o t change w h e n a n i m a l s were i n j e c t e d i n t r a v e n o u s l y with a pulse i n j e c t i o n of Ch. However, Dross a n d Kewitz (1972) f o u n d t h a t blood b o r n e Ch p e n e t r a t e s easily t h r o u g h the blood b r a i n barrier of rats even t h o u g h the concent r a t i o n g r a d i e n t is from b r a i n to blood. Since c u r r e n t m e t h o d s to measure the t u r n o v e r rate of ACh i n b r a i n tissue after i n j e c t i o n of labeled
Send of]print requests to: D. L. Cheney, Laboratory of Preelinieal Pharmacology, National Inst. of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A. 7*
100
G. gacagni et al.
Ch ( J e n d e n et al., 1974; Saelens et a[., 1973; S c h u b e r t h et al., 1970; Dross a n d Kewitz, 1966) assume s t e a d y state a n d calculate t u r n o v e r rate b y a p p l y i n g principles of s t e a d y - s t a t e kinetics to the change with t i m e of Ch a n d ACh specific activity, it is i m p o r t a n t to d e t e r m i n e whether or n o t the i n t r a v e n o u s i n j e c t i o n of Ch alters the Ch or ACh c o n t e n t i n various regions of r a t brain.
Methods In our experiments ~r Spr~gue-Dawley rats (Zivic-Miller, Allison Park, Pa.) weighing between 120--150 g were used. The animals were kept at constant temperature (23~176 with alternating light/dark periods of 14 and 10 hrs, respectively. The experiments were carried out in the morning in order to eliminate fluctuations in Ch and ACh content due to diurnal variations (Hanin et al., 1970). Compounds were dissolved in 0.90/0 NaC1. The deuterated compounds, Ch-~Hr or Ch-D~ [N+(CH3)~ C~DaOH Br], ACh-2H~ or ACh-Da [N+(CH3)a C2D~OC (0) CH a Br], Ch-2H9 or Ch-D9 [N+(CD3)3 C2H~OH Br], and ACh-~H9 or ACh-D 9 [N+(CD3)3 C~H~OC (0) Ctta Br] were obtained from Merck, Sharp and Dohme, Quebec, Canada. Ch-Da (1, 5 or 25 ~moles kg -1 mill-1) was infused through the rat tail vein for 6 min at a constant rate (0.2 ml min-1). During the infusion, rats were not anesthetized but were restrained in plastic cages. At the end of the infusion, there was no evidence of sedation. Tim rats were then killed by exposing their heads for 2 see to a focused beam of microwave radiation (2.0 kW; 2.45 GHz, 75 mW cm2) (Stavinoh~ and Weinstraub, 1974; Guidotti et al., 1974; Racagni et al., 1974). Koslow et al. (1974) have shown that this killing procedure stabilizes the brain concentration of Ch and ACh and allows dissection and assay of ACh in brain nuclei. The brain regions were dissected and the ACh and Ch extracted according to the method of Hanin et al. (1973) as modified by Cheney et al. (1975a). To measure plasma concentrations of Ch, animals were decapitated without exposing them to microwave radiation in order to avoid hemolysis. Preparation of extracts for GC-MS were described previously (Cheney et al., 1975b). Concentrations of ACh, Ch, ACh-Da, ACh-D 9 were measured with a quadrupole gas chromatograph-mass spectrometer (GC-~IS) (Finnigan 3000). The conditions for gas chromatography were 30 ml/min of helium flow; flash heater 240~ column 145~ The derivatives were injected into an 8 foot glass column (3 mm, i.d.) packed with 28~ Penwalt 223 and 40/0 KOH on Gas Chrom R. Ch-D9 and ACh-D 9 were used as internal standards. Ion fragments with m/e of 58, 60 and 64 were selected for mass fragmcntographic determination of Ch and ACh as described by ttanin and Schuberth (1974) and Hammer et al. (1968).
Results The c o n c e n t r a t i o n of e n d o g e n o u s p l a s m a Ch did n o t change significantly i n rats infused with Ch-D a (Fig. 1). However, t o t a l Ch (endogenous Ch plus Ch-Dd) increased significantly w h e n rates of either 5 ~moles kg -1 m i n -1 or 25 ~moles kg -1 m i n -~ (Fig. 1) were infused. W i t h the infusion of 5 ~moles kg -1 m i n -1 of Ch-D 4 the plasma c o n t e n t of Ch (Ch -~ Ch-Dd) a l m o s t d o u b l e d a t 2 m i n of i n f u s i o n a n d this level was m a i n t a i n e d for the following 6 rain. I n contrast, w h e n 25 ~moles kg -1 rain -1 were
Steady-State Concentrations of Choline and Aeetyloholine I00 90 80
101
A
70 =
60 5r 40 :30 o
20
15
I00 MINUTES
Fig. 1. Nanomoles of endogenous Ch (open symbols) and endogenous Ch plus Ch-Dt (closed symbols) per ml plasma following intravenous constant rate infusion of Ch-D4. Panel A indicates infusion of 5 ~moles kg-z rain -1. Panel B indicates infusion of 25 ~moles kg -1 min -1. Data represent mean 4- standard error of 4--6 determinations
infused the plasma c o n t e n t of Ch-D 4 continuted to increase for at least 8 rain. Table 1 shows the concentration of Ch, ACh, Ch-D 4 a n d ACh-D 4 in striatum a n d occipital cortex after 6 rain of c o n s t a n t rate infusion of 1, 5, or 25 ~zmoles kg -1 min -1 of Ch-D 4. Ch concentration increased significantly in b o t h occipital cortex a n d s t r i a t u m after a 6 rain infusion of 5 or 25 ~zmoles kg -1 min -1 of Oh. N o increase was o b t a i n e d with an infusion rate of 1 [zmole kg -1 Inin -1. Total ACh concentration was significantly increased in occipital cortex after 5 a n d 25 [zmoles kg -1 min -1 Ch-D 4 and in s t r i a t u m after 25 ~zmoles kg -1 rain -1 Ch-D,. To verify whether or n o t these changes observed with Ch-D 4 were due to an isotopic effect, we injected a comparable concentration of Ch (200 ~zmoles kg -1) i n t r a v e n o u s l y as a single pulse. The d a t a of Table 2 show t h a t a single pulse injection of a large dose of Ch caused a tremendous increase in the plasma Ch c o n t e n t 30 see after the injection. Moreover, there was a 250/0 increase in ACh concentration a n d a 147~ increase in Oh concentration in s t r i a t u m 30 see after injection. W i t h i n 2 rain the concentrations of ACh a n d Ch h a d r e t u r n e d to basal values.
i 4.5 ~ 2.6 :j:: 3.4 • 2.3
-0.80 ::t: 0.13 2.1 :E 0.27 7.0 q- 0.47
-0.30 =]= 0.027 1.3 ~ 0.067 2.1 =]=0.067
ACh-D~ (nmoles g-l)
64 71 63 77
:J: 4.5 • 2.5 ::]: 3.3 ~= 1.9 b
12 ~= 0.80 13 i 0.54 14 i 0.40 b 15~=0.67 b
Total ACh ~ ACh-D~ (nmoles g-X)
39 46 48 54
• 2.8 ~ 4.9 :J: 1.8 b • 2.5b
28 • 3,5 28 i 1,7 35 i 2.5 28•
Ch (nmoles g-X)
-0.8 :J:: 0.2 4.5 • 1.2 14 • 1.3
-1,6 -4- 0.5 6.1 -4- 1.7 16 =]=0.4
Ch-D 4 (nmoles g-X)
a Ch-D~ was infused at a constant rate for 6 min. D a t a represent m e a n -4- s t a n d a r d error of 6 determinations. b p < 0.05 when compared to animals infused with physiologic saline.
None 1 ~molc kg-1 min -I 5 ~molc kg-1 rain -1 25 ~mole kg -x min -x
~triatum
64 70 61 70
12 ~= 0.80 13 ~= 0.54 13 j= 0.47 13~=0.67
None 1 ~mole kg -1 min -1 5 ~mole kg -1 min -1 2 5 ~ m o l e k g - l m i n -1
Occipital cortex
ACh (nmoles g-l)
Amount of Ch-D~ infused
39 47 53 68
• • • ~
2.8 4.8 2.0 b 3.7 u
28 ~= 3.5 29 =L 1.9 41 • 3.1 b 44• b
Total Ch ~- Ch-D 4 (nmoles g-l)
Table 1. ACh and Ch concentrations in occipital cortex and s t r i a t u m of rats receiving a constant rate infusion of Ch-D~ ~
Steady-State Concentrations of Choline and Aeetylcholine
103
Table 2. Acetylcholine and choline concentrations in rat striatum at various times following a rapid intravenous injection of choline (200 tzmoles kg-1) Time after injection (min)
Choline (nmoles m1-1 plasma)
Acetylcholine (nmoles g-l)
Choline (nmoles g-l)
0 0.5 2.0
13 4- 1.2 1600 • 97 b --
56 4- 4.0 70 • 7.9 b 64 • 5.1
32 • 4.5 79 • 4.8 b 42 4- 1.0
a Data are expressed as mean 4- standard error of 5 determinations. b p < 0.01.
Discussion The data reported in Fig. 1 show that only a small portion of the 5 txmoles kg -1 rain -I of Ch-D 4 infused intravenously at constant rate was retained in plasma after 2 rain of infusion. This amount did not increase further when the infusion was prolonged up to 8 min. I t has been shown that liver, kidney and other tissues can rapidly remove Ch from the blood (Gardiner and Paton, 1972). Our results demonstrate that the brain Ch concentration was augmented when plasma Ch content was increased during the constant rate of infusion of 25 fzmoles kg -1 rain -1 of Ch-D4. During the infusion of this dose of Ch-D 4 at constant rate the increase in the plasma concentrations of Ch was proportional to the infusion time as shown in Fig. 1. Following the infusion of 5 or 25 ~moles kg -1 rain -1 at constant rate for 6 rain there was a significant increase in the total Ch content of striatum and cortex. This suggests that the brain content of Ch increased when the plasma concentration of Ch was increased from 13 nmoles m1-1 to 23 nmoles m1-1. The increased content of Ch in cortex was associated with an increase of ACh content in cortex (Table 1). I n striatum, however the increased ACh content was observed only after infusion of 25 ~moles kg -1 rain -1. An example of the regulation of ACh concentrations at non-steady state conditions is given in Table 2. These data show that after a single intravenous pulse injection of 200 ~molcs kg -1 of Ch the concentrations of both Ch and ACh increased 30 sec after injection but they returned to normal within 2 rain. Since 2--30/0 of the brain weight is composed of blood (Kewitz, personal communication) and since the blood contains 1600 nmoles m1-1 30 see after the pulse injection of 200 ~moles kg -1 of Ch there would be 32--48 nmoles g-1 of Ch contributed by the blood within the tissue. This amount becomes relevant when the brains are fixed by microwave irradiation because the blood is coagulated and remains trapped in the tissue. Thus, the increase of striatal Ch is due to the
104
G. Racagni et al.
plasma Ch present within the tissue. However, the increased ACh cont e n t in striatum occurs because of an enhanced synthesis of ACh. H a u b rich et al. (1974) reported no change in the ACh and Ch c o n t e n t of guinea pig brain 2 rain after pulse injection of 200 ~moles/kg -1 of Ch. However, our d a t a show t h a t 2 min m a y n o t be an appropriate time to detect the p e r t u r b a t i o n of the s t e a d y state. During the intravenous infusion of Ch-D 4 at c o n s t a n t rate even m o d e r a t e doses of Ch (5 ~zmoles kg -1 rain -1) caused the concentration of Ch in cortex or striatum to increase b y a b o u t 40 ~ after 6 min of infusion. I t appears t h a t 1 ~zmole kg -1 rain -1 of Ch would be close to the maximal infusion rate t h a t could be used for t u r n o v e r experiments using deut e r a t e d Ch as the label. The present studies have confirmed t h a t the brain ACh c o n t e n t increases when Ch-D 4 concentrations in plasma are elevated. This was n o t an isotopic effect because it has been observed even with the n o n v a r i a n t form of Ch. Moreover, the infusion of Ch-D 4 increased the striatal c o n t e n t of endogenous Ch. I n addition, the d a t a presented reaffirms t h a t when pulse injection of radioactive Ch is used to measure ACh t u r n o v e r rate the maintenance of s t e a d y state conditions m u s t be shown b y measuring brain Ch and ACh at v e r y early times following the pulse injection. Acknowledgement. We wish to acknowledge the expert technical assistance of Mr. Richard Shirasawa,
References Cheney, D. L., Costa, E., Hanin, I,, Trabucchi, ~., Wang, C. T. : Application of principles of Steady State Kinetics to the in vivo estimation of acetylcholine turnover rate in mouse brain. J. Pharmacol. exp. Ther. 192, 288--296 (1975a) Cheney, D. L., LeFevre, H., Racagni, G.: Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brain. Neuropharmacology (in press, 1975b) Dross, K., Kewitz, H. : Der Einbau yon i.v. zugefiihrtem Cholin in das Acetylcholin des Gehirns. Naunyn-Schmiedeberg's Arch. Pharmacol. 255, 10--ll (1966) Dross, K., Kewitz, H. : Concentration and origin of choline in rat brain. NaunynSchmiedeberg's Arch. Pharmacol. 274, 91--106 (1972) Gardiner, J. E., Patch, W. D. M. : The control of the plasma choline concentration in the cut. J. Physiol. (Lond.) 227, 71--86 (1972) Guidotti, A., Cheney, D. L., Trabucchi, M,, Doteuchi, M., Wang, C., Hawkins, R.A. : Focussed microwave radiation: a technique to minimize post mortem changes of cyclic nucleotides, DOPA and choline and to preserve brain morphology. Neuropharmacology 13, 1115--1122 (1974) Hammar, C. G., ttanin, I., Holmstedt, B., Kitz, R. J., Jenden, D. J., Karlen, B.: Identification of acetylcholine in fresh rat brain by combined gas chromatography-mass spectrometry. Nature (Lond.) 220, 915--917 (1968) Hanin, I., Cheney, D.L., Trabucchi, lCI., Massarelli, R., Wang, C.T., Costa, E.: Application of principles of steady-state kinetics to measure acetyleholine turnover rate in rat salivary glands. Effect of deafferentiation and duct ligation. J. Pharmaeol. exp. Ther. 187, 68--77 (1973)
Steady-Sta~e Concentrations of Choline and Aeetylcholine
105
Hanin, L, Massarelli, R., Costa, E.: Acetyleholine concentrations in rat brain: Diurn~I oseillation. Science 170, 341--342 (1970) Hanin, L, Sehuberth, J.: Labelling of acetyleholme in the rat brain of mice fed on a diet containing deuterium labelled choline: studies utilizing gas chromatographymass spectrometry. J. Neuroehem. 23, 819--824 (1974) Haubrieh, D. R., Wedeking, 1~. W., Wang, P. F. L.: Increase in tissue concentration of acetylcholine in guinea pigs in rive induced by administration of choline. Life Sci. 14, 921--927 (t974) Jenden, D. J., Choi, L., Silverman, ~. W., Steinborn, J. A., Roch, M., Booth, g. A.: Aeetyleholine turnover estimation in brain by gas chromatography-mass spectrometry. Life Sei. 14, 55-63 (1974) Koslow, S. H., l~aeagni, G., Cos~a, E.: ~fass fragmentographie measurement of norepinephrine, dopamine, sero~onin and acetylehotine in seven discrete nuclei of the rat teldiencephalon. Neuroph~rmaeology 13, 1123 - 1130 (1974) Racagni, G., Cheney, D. L., Trabucchi, M., Wang, C,, Costa, E.: l~easurement os Acetyleholine turnover rate in discrete areas of rat brain. Life Sei. 15, 1961-- 1976 (1974) Saelens, J. K., Simke, J. P., Allen, M. P., Conroy, C. A., Some of the dynamics of choline, and acetylcholine metabolism in rat brain. Arch. int. I)harm~col. ~08, 305--312 (1973) Schuberth, J., Sparf, B., Sundwall, A. : On the turnover of acetylcholine in nerve endings of mouse brain in rive. J. Neurochem. 17, 461--468 (1979) St~vi~oha, W. B., Weintraub, S. T.: Choline eon?~ent of rat brain. Science 183, 964- 965 (1974)
Short Communication Steady.State Concentrations of Choline and Acetylcholine in Rat Brain Parts during a Constant Rate Infusion of Deuterated Choline G. Racagni, M. Trabucchi, a n d D. L. Cheney Laboratory of Preclinical Pharmacology, National Institute of i~[ental Health, Saint Elizabeths Hospital, Washington, D.C. Received March 7 / Accepted June 11, 1975
Summary. An intravenous infusion of deuteratod choline at constant rate for 6 rain (5 or 25 ~zmoleskg -1 rain-1) significantly increases the concentration of choline in plasma,, occipital cortex and striatum. Both 5 and 25 ~moles kg -~ rain-1 increase the concentration of acetylcholine in cortex but only 25 [zmoles kg-1 min-1 increases the acetyleholine content in striatum. In contrast, 1 [zrnolekg -1 rain-1 does not change the choline or acetylcholine content in cortex or striatum. A single pulse injection of choline (200 tzmoles kg-1) causes a significant increase in the concentration of choline in striatum 30 see following injection. The choline content returns to normal values within 2 rain. These studies show that when a pulse injection of a non-tracer dose of radioactive choline is used to measure brain aeetylcholine turnover rate the maintenance of steady state must be verified within seconds after the pulse injection of radioactive choline. When constant infusion of deuterated choline is used to measure turnover rate of acetylcholine in the brain of rats, a dose of I ~Lmolekg-1 rain-1 appears to be a maximal infusion rate. Key words: Choline -- Acetylcholine -- Infusion -- Striatum -- Occipital Cortex -- Mass Fragmentography.
Introduction R e c e n t studies i n whole b r a i n of g u i n e a pigs ( H a u b r i c h et al., 1974) a n d mice ( J e n d e n et al., 1974) suggested t h a t the s t e a d y - s t a t e concent r a t i o n s of choline (Ch) a n d acetylcholine (ACh) did n o t change w h e n a n i m a l s were i n j e c t e d i n t r a v e n o u s l y with a pulse i n j e c t i o n of Ch. However, Dross a n d Kewitz (1972) f o u n d t h a t blood b o r n e Ch p e n e t r a t e s easily t h r o u g h the blood b r a i n barrier of rats even t h o u g h the concent r a t i o n g r a d i e n t is from b r a i n to blood. Since c u r r e n t m e t h o d s to measure the t u r n o v e r rate of ACh i n b r a i n tissue after i n j e c t i o n of labeled
Send of]print requests to: D. L. Cheney, Laboratory of Preelinieal Pharmacology, National Inst. of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A. 7*
100
G. gacagni et al.
Ch ( J e n d e n et al., 1974; Saelens et a[., 1973; S c h u b e r t h et al., 1970; Dross a n d Kewitz, 1966) assume s t e a d y state a n d calculate t u r n o v e r rate b y a p p l y i n g principles of s t e a d y - s t a t e kinetics to the change with t i m e of Ch a n d ACh specific activity, it is i m p o r t a n t to d e t e r m i n e whether or n o t the i n t r a v e n o u s i n j e c t i o n of Ch alters the Ch or ACh c o n t e n t i n various regions of r a t brain.
Methods In our experiments ~r Spr~gue-Dawley rats (Zivic-Miller, Allison Park, Pa.) weighing between 120--150 g were used. The animals were kept at constant temperature (23~176 with alternating light/dark periods of 14 and 10 hrs, respectively. The experiments were carried out in the morning in order to eliminate fluctuations in Ch and ACh content due to diurnal variations (Hanin et al., 1970). Compounds were dissolved in 0.90/0 NaC1. The deuterated compounds, Ch-~Hr or Ch-D~ [N+(CH3)~ C~DaOH Br], ACh-2H~ or ACh-Da [N+(CH3)a C2D~OC (0) CH a Br], Ch-2H9 or Ch-D9 [N+(CD3)3 C2H~OH Br], and ACh-~H9 or ACh-D 9 [N+(CD3)3 C~H~OC (0) Ctta Br] were obtained from Merck, Sharp and Dohme, Quebec, Canada. Ch-Da (1, 5 or 25 ~moles kg -1 mill-1) was infused through the rat tail vein for 6 min at a constant rate (0.2 ml min-1). During the infusion, rats were not anesthetized but were restrained in plastic cages. At the end of the infusion, there was no evidence of sedation. Tim rats were then killed by exposing their heads for 2 see to a focused beam of microwave radiation (2.0 kW; 2.45 GHz, 75 mW cm2) (Stavinoh~ and Weinstraub, 1974; Guidotti et al., 1974; Racagni et al., 1974). Koslow et al. (1974) have shown that this killing procedure stabilizes the brain concentration of Ch and ACh and allows dissection and assay of ACh in brain nuclei. The brain regions were dissected and the ACh and Ch extracted according to the method of Hanin et al. (1973) as modified by Cheney et al. (1975a). To measure plasma concentrations of Ch, animals were decapitated without exposing them to microwave radiation in order to avoid hemolysis. Preparation of extracts for GC-MS were described previously (Cheney et al., 1975b). Concentrations of ACh, Ch, ACh-Da, ACh-D 9 were measured with a quadrupole gas chromatograph-mass spectrometer (GC-~IS) (Finnigan 3000). The conditions for gas chromatography were 30 ml/min of helium flow; flash heater 240~ column 145~ The derivatives were injected into an 8 foot glass column (3 mm, i.d.) packed with 28~ Penwalt 223 and 40/0 KOH on Gas Chrom R. Ch-D9 and ACh-D 9 were used as internal standards. Ion fragments with m/e of 58, 60 and 64 were selected for mass fragmcntographic determination of Ch and ACh as described by ttanin and Schuberth (1974) and Hammer et al. (1968).
Results The c o n c e n t r a t i o n of e n d o g e n o u s p l a s m a Ch did n o t change significantly i n rats infused with Ch-D a (Fig. 1). However, t o t a l Ch (endogenous Ch plus Ch-Dd) increased significantly w h e n rates of either 5 ~moles kg -1 m i n -1 or 25 ~moles kg -1 m i n -~ (Fig. 1) were infused. W i t h the infusion of 5 ~moles kg -1 m i n -1 of Ch-D 4 the plasma c o n t e n t of Ch (Ch -~ Ch-Dd) a l m o s t d o u b l e d a t 2 m i n of i n f u s i o n a n d this level was m a i n t a i n e d for the following 6 rain. I n contrast, w h e n 25 ~moles kg -1 rain -1 were
Steady-State Concentrations of Choline and Aeetyloholine I00 90 80
101
A
70 =
60 5r 40 :30 o
20
15
I00 MINUTES
Fig. 1. Nanomoles of endogenous Ch (open symbols) and endogenous Ch plus Ch-Dt (closed symbols) per ml plasma following intravenous constant rate infusion of Ch-D4. Panel A indicates infusion of 5 ~moles kg-z rain -1. Panel B indicates infusion of 25 ~moles kg -1 min -1. Data represent mean 4- standard error of 4--6 determinations
infused the plasma c o n t e n t of Ch-D 4 continuted to increase for at least 8 rain. Table 1 shows the concentration of Ch, ACh, Ch-D 4 a n d ACh-D 4 in striatum a n d occipital cortex after 6 rain of c o n s t a n t rate infusion of 1, 5, or 25 ~zmoles kg -1 min -1 of Ch-D 4. Ch concentration increased significantly in b o t h occipital cortex a n d s t r i a t u m after a 6 rain infusion of 5 or 25 ~zmoles kg -1 min -1 of Oh. N o increase was o b t a i n e d with an infusion rate of 1 [zmole kg -1 Inin -1. Total ACh concentration was significantly increased in occipital cortex after 5 a n d 25 [zmoles kg -1 min -1 Ch-D 4 and in s t r i a t u m after 25 ~zmoles kg -1 rain -1 Ch-D,. To verify whether or n o t these changes observed with Ch-D 4 were due to an isotopic effect, we injected a comparable concentration of Ch (200 ~zmoles kg -1) i n t r a v e n o u s l y as a single pulse. The d a t a of Table 2 show t h a t a single pulse injection of a large dose of Ch caused a tremendous increase in the plasma Ch c o n t e n t 30 see after the injection. Moreover, there was a 250/0 increase in ACh concentration a n d a 147~ increase in Oh concentration in s t r i a t u m 30 see after injection. W i t h i n 2 rain the concentrations of ACh a n d Ch h a d r e t u r n e d to basal values.
i 4.5 ~ 2.6 :j:: 3.4 • 2.3
-0.80 ::t: 0.13 2.1 :E 0.27 7.0 q- 0.47
-0.30 =]= 0.027 1.3 ~ 0.067 2.1 =]=0.067
ACh-D~ (nmoles g-l)
64 71 63 77
:J: 4.5 • 2.5 ::]: 3.3 ~= 1.9 b
12 ~= 0.80 13 i 0.54 14 i 0.40 b 15~=0.67 b
Total ACh ~ ACh-D~ (nmoles g-X)
39 46 48 54
• 2.8 ~ 4.9 :J: 1.8 b • 2.5b
28 • 3,5 28 i 1,7 35 i 2.5 28•
Ch (nmoles g-X)
-0.8 :J:: 0.2 4.5 • 1.2 14 • 1.3
-1,6 -4- 0.5 6.1 -4- 1.7 16 =]=0.4
Ch-D 4 (nmoles g-X)
a Ch-D~ was infused at a constant rate for 6 min. D a t a represent m e a n -4- s t a n d a r d error of 6 determinations. b p < 0.05 when compared to animals infused with physiologic saline.
None 1 ~molc kg-1 min -I 5 ~molc kg-1 rain -1 25 ~mole kg -x min -x
~triatum
64 70 61 70
12 ~= 0.80 13 ~= 0.54 13 j= 0.47 13~=0.67
None 1 ~mole kg -1 min -1 5 ~mole kg -1 min -1 2 5 ~ m o l e k g - l m i n -1
Occipital cortex
ACh (nmoles g-l)
Amount of Ch-D~ infused
39 47 53 68
• • • ~
2.8 4.8 2.0 b 3.7 u
28 ~= 3.5 29 =L 1.9 41 • 3.1 b 44• b
Total Ch ~- Ch-D 4 (nmoles g-l)
Table 1. ACh and Ch concentrations in occipital cortex and s t r i a t u m of rats receiving a constant rate infusion of Ch-D~ ~
Steady-State Concentrations of Choline and Aeetylcholine
103
Table 2. Acetylcholine and choline concentrations in rat striatum at various times following a rapid intravenous injection of choline (200 tzmoles kg-1) Time after injection (min)
Choline (nmoles m1-1 plasma)
Acetylcholine (nmoles g-l)
Choline (nmoles g-l)
0 0.5 2.0
13 4- 1.2 1600 • 97 b --
56 4- 4.0 70 • 7.9 b 64 • 5.1
32 • 4.5 79 • 4.8 b 42 4- 1.0
a Data are expressed as mean 4- standard error of 5 determinations. b p < 0.01.
Discussion The data reported in Fig. 1 show that only a small portion of the 5 txmoles kg -1 rain -I of Ch-D 4 infused intravenously at constant rate was retained in plasma after 2 rain of infusion. This amount did not increase further when the infusion was prolonged up to 8 min. I t has been shown that liver, kidney and other tissues can rapidly remove Ch from the blood (Gardiner and Paton, 1972). Our results demonstrate that the brain Ch concentration was augmented when plasma Ch content was increased during the constant rate of infusion of 25 fzmoles kg -1 rain -1 of Ch-D4. During the infusion of this dose of Ch-D 4 at constant rate the increase in the plasma concentrations of Ch was proportional to the infusion time as shown in Fig. 1. Following the infusion of 5 or 25 ~moles kg -1 rain -1 at constant rate for 6 rain there was a significant increase in the total Ch content of striatum and cortex. This suggests that the brain content of Ch increased when the plasma concentration of Ch was increased from 13 nmoles m1-1 to 23 nmoles m1-1. The increased content of Ch in cortex was associated with an increase of ACh content in cortex (Table 1). I n striatum, however the increased ACh content was observed only after infusion of 25 ~moles kg -1 rain -1. An example of the regulation of ACh concentrations at non-steady state conditions is given in Table 2. These data show that after a single intravenous pulse injection of 200 ~molcs kg -1 of Ch the concentrations of both Ch and ACh increased 30 sec after injection but they returned to normal within 2 rain. Since 2--30/0 of the brain weight is composed of blood (Kewitz, personal communication) and since the blood contains 1600 nmoles m1-1 30 see after the pulse injection of 200 ~moles kg -1 of Ch there would be 32--48 nmoles g-1 of Ch contributed by the blood within the tissue. This amount becomes relevant when the brains are fixed by microwave irradiation because the blood is coagulated and remains trapped in the tissue. Thus, the increase of striatal Ch is due to the
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plasma Ch present within the tissue. However, the increased ACh cont e n t in striatum occurs because of an enhanced synthesis of ACh. H a u b rich et al. (1974) reported no change in the ACh and Ch c o n t e n t of guinea pig brain 2 rain after pulse injection of 200 ~moles/kg -1 of Ch. However, our d a t a show t h a t 2 min m a y n o t be an appropriate time to detect the p e r t u r b a t i o n of the s t e a d y state. During the intravenous infusion of Ch-D 4 at c o n s t a n t rate even m o d e r a t e doses of Ch (5 ~zmoles kg -1 rain -1) caused the concentration of Ch in cortex or striatum to increase b y a b o u t 40 ~ after 6 min of infusion. I t appears t h a t 1 ~zmole kg -1 rain -1 of Ch would be close to the maximal infusion rate t h a t could be used for t u r n o v e r experiments using deut e r a t e d Ch as the label. The present studies have confirmed t h a t the brain ACh c o n t e n t increases when Ch-D 4 concentrations in plasma are elevated. This was n o t an isotopic effect because it has been observed even with the n o n v a r i a n t form of Ch. Moreover, the infusion of Ch-D 4 increased the striatal c o n t e n t of endogenous Ch. I n addition, the d a t a presented reaffirms t h a t when pulse injection of radioactive Ch is used to measure ACh t u r n o v e r rate the maintenance of s t e a d y state conditions m u s t be shown b y measuring brain Ch and ACh at v e r y early times following the pulse injection. Acknowledgement. We wish to acknowledge the expert technical assistance of Mr. Richard Shirasawa,
References Cheney, D. L., Costa, E., Hanin, I,, Trabucchi, ~., Wang, C. T. : Application of principles of Steady State Kinetics to the in vivo estimation of acetylcholine turnover rate in mouse brain. J. Pharmacol. exp. Ther. 192, 288--296 (1975a) Cheney, D. L., LeFevre, H., Racagni, G.: Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brain. Neuropharmacology (in press, 1975b) Dross, K., Kewitz, H. : Der Einbau yon i.v. zugefiihrtem Cholin in das Acetylcholin des Gehirns. Naunyn-Schmiedeberg's Arch. Pharmacol. 255, 10--ll (1966) Dross, K., Kewitz, H. : Concentration and origin of choline in rat brain. NaunynSchmiedeberg's Arch. Pharmacol. 274, 91--106 (1972) Gardiner, J. E., Patch, W. D. M. : The control of the plasma choline concentration in the cut. J. Physiol. (Lond.) 227, 71--86 (1972) Guidotti, A., Cheney, D. L., Trabucchi, M,, Doteuchi, M., Wang, C., Hawkins, R.A. : Focussed microwave radiation: a technique to minimize post mortem changes of cyclic nucleotides, DOPA and choline and to preserve brain morphology. Neuropharmacology 13, 1115--1122 (1974) Hammar, C. G., ttanin, I., Holmstedt, B., Kitz, R. J., Jenden, D. J., Karlen, B.: Identification of acetylcholine in fresh rat brain by combined gas chromatography-mass spectrometry. Nature (Lond.) 220, 915--917 (1968) Hanin, I., Cheney, D.L., Trabucchi, lCI., Massarelli, R., Wang, C.T., Costa, E.: Application of principles of steady-state kinetics to measure acetyleholine turnover rate in rat salivary glands. Effect of deafferentiation and duct ligation. J. Pharmaeol. exp. Ther. 187, 68--77 (1973)
Steady-Sta~e Concentrations of Choline and Aeetylcholine
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Hanin, L, Massarelli, R., Costa, E.: Acetyleholine concentrations in rat brain: Diurn~I oseillation. Science 170, 341--342 (1970) Hanin, L, Sehuberth, J.: Labelling of acetyleholme in the rat brain of mice fed on a diet containing deuterium labelled choline: studies utilizing gas chromatographymass spectrometry. J. Neuroehem. 23, 819--824 (1974) Haubrieh, D. R., Wedeking, 1~. W., Wang, P. F. L.: Increase in tissue concentration of acetylcholine in guinea pigs in rive induced by administration of choline. Life Sci. 14, 921--927 (t974) Jenden, D. J., Choi, L., Silverman, ~. W., Steinborn, J. A., Roch, M., Booth, g. A.: Aeetyleholine turnover estimation in brain by gas chromatography-mass spectrometry. Life Sei. 14, 55-63 (1974) Koslow, S. H., l~aeagni, G., Cos~a, E.: ~fass fragmentographie measurement of norepinephrine, dopamine, sero~onin and acetylehotine in seven discrete nuclei of the rat teldiencephalon. Neuroph~rmaeology 13, 1123 - 1130 (1974) Racagni, G., Cheney, D. L., Trabucchi, M., Wang, C,, Costa, E.: l~easurement os Acetyleholine turnover rate in discrete areas of rat brain. Life Sei. 15, 1961-- 1976 (1974) Saelens, J. K., Simke, J. P., Allen, M. P., Conroy, C. A., Some of the dynamics of choline, and acetylcholine metabolism in rat brain. Arch. int. I)harm~col. ~08, 305--312 (1973) Schuberth, J., Sparf, B., Sundwall, A. : On the turnover of acetylcholine in nerve endings of mouse brain in rive. J. Neurochem. 17, 461--468 (1979) St~vi~oha, W. B., Weintraub, S. T.: Choline eon?~ent of rat brain. Science 183, 964- 965 (1974)
