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Uptake and metabolism of cyclic AMP in rabbit choroid plexus in vitro
Neuropharmacology,
1977, 16, 135-141.
Pergamon
Press.
Printed
in Gt. Britain.
UPTAKE AND METABOLISM OF CYCLIC AMP IN RABBIT CHOROID PLEXUS IN VITRO* R. HAMMERS,P. CLARJZNBACH, T. LINDL~ and H. CRAMER Department of Neurology, University of Freiburg, Freiburg, FRG (Accepted 22 June 1976) Summary-The uptake and fate of labelled cyclic nucleotides in isolated rabbit choroid plexus were studied in vitro. Radioactivity accumulated almost linearily for 240 min, reaching tissue/medium ratios of 89 f 2.4 for cyclic AMP and 83 f 2.5 for cyclic GMP. The uptake process was saturable at substrate concentrations ranging from l.OpM to 10mM. The uptake of [3H]-cyclic AMP was significantly inhibited by lowering of temperature, anoxia, ouabain and omission of sodium as well as by cyclic GMP, S-AMP, probenecid and several other organic acids but not by neutral or basic compounds. Thin layer chromatographic identification of the intracellular radioactivity revealed that after 10 min incubation less than 10% was attributable to cyclic AMP itself, whereas about 45% was associated with ATP + ADP and 2% with the acid insoluble fraction of the choroid plexus. Inhibition of phosphodiesterase activity by theophylline increased the intracellular cyclic AMP fraction of total radioactivity accumulated, but decreased the tissue/medium ratio by about two thirds. The finding of a cyclic AMP generating system sensitive to histamine and prostaglandin E,, but not to noradrenaline, in the choroid plexus suggests a functional role of cyclic AMP in this tissue.
The choroid plexuses of the ventricular cavities are the major sites of production of ccrebrospinal fluid (CSF). (BERING, 1955; ROUGEMONT, AMES, NESBETT and HOFMANN, 1960). More recently data have accumulated suggesting that the choroid plexuses, as well as other parts of the ventricular lining and the spinal pia mater, have an important function in removing metaholites which have entered the CSF compartment from the brain tissue (CSERR, 1971). For the monoamine metabolites homovanillic acid (HVA) and 5hydroxyindoleacetic acid (5HIAA) as well as for a number of foreign compounds and drugs, the transport from CSF to choroidal venous blood has been studied extensively suggesting the existence of mediated transport with several distinct carriers (P~PPENHEIMER,HEISEYand JORDAN, 1961; CSERR and VAN DYKE, 1971. Another brain metabolite, 3’,5’-cyclit adenosine monophosphate (cyclic AMP), occurs in mammalian CSF in concentrations similar to those of the plasma, (ROBISON, COPPEN, WHYBROW and F’RANGE, 1970; CRAMER, GOODWIN, POST and BUNNEY, 1972a) but concentrations vary independently in both compartments (CRAMER and LINDL, 1972; CRAMER, NG and CHASE, 197217). Similarly to the acidic metabolites of monoamines, cyclic AMP ac-
cumulates in CSF after the administration of probenecid (CRAMER, GOODWIN, POST and BUNNEY, 1972a), a drug which inhibits carrier mediated transport of organic anions in the plexus (GULDBERG,ASHCROFT and CRAWFORD,1966; NEFF, TOZER and BRODIE, 1967; FORN 1972). From isolated intact nervous tissue cyclic AMP is released in considerable amounts in relationship to its intracellular formation (CRAMER and LINDL, 1974), and it can be hypothesized that extracellular extrusion, passage into the CSF and transport out of CSF via the choroid plexus and pia mater might be an additional mechanism of inactivation of this cyclic nucleotide in the central nervous system. In order to test further this hypothesis we studied the uptake and fate of labelled cyclic nucleotides in the isolated choroid plexus of the rabbit in vitro. METHODS Randomly bred male rabbits of 2-3.5 kg body weight were killed by cervical dislocation. After rapid removal of the brain the lateral ventricles were opened, their choroid plexuses excised and transferred into chilled Krebs-Henseleit solution. Pial tissue was stripped from the convexity of the hemispheres and treated similarly.
* Supported by the Deutsche Forschungsgemeinschaft (SFB 70). Work presented in this paper is part of a thesis to be submitted to the Medical Faculty of the University of Preiburg (R.H.). t Resent address: Department of Biology, University of Konstanz, D-775 Konst&z. Reprint requests to: Professor H. Cramer. Department of Neurology, University of Freiburg, D-7800 Ereiburg, FRG. -Key words: adenosine 3$monophosphate, guanosine 3,5-monophosphate, cerebrospinal fluid, choroid plexus, tramport, phosphodiesterase, theophylline, histamine.
Uptake of cyclic nucleotides Each incubation tube contained 2 plexuses in 4ml of Krebs-Henseleit solution aerated with 95% O2 and 5% COz (pH 7.4). Unlahelled cyclic AMP or 3’,5’-cyclit guanosine monophosphate (cyclic GMP) in various concentrations and labelled nucleotide (0.5 &i/ml; specific activities: 27.5 Ci/mmol for cyclic AMP and 15.0Ci/mmol for cyclic GMP, both from Amersham Ltd.) were added.
135
R. HAMMERS,P. CLARENBACH, T. LINDLand H. CRAMER
136
After incubations at 37°C from 10 to 24Omin, the plexuses were removed from the medium, washed, transferred into scintillation vials and dissolved with Soluene TM 100. The hydrolysate was neutralized with concentrated HCl for higher counting efficiency, dissolved in butyl-PBD-toluol scintillation fluid and counted. Radioactivity was also counted in the media. After correction for quenching and tissue/water ratio the results were expressed as tissue/medium ratio. Isolation ofcyclic AMP metabolites by thin layer chromatography
After incubation the plexuses were homogenized in 6% trichloric acid and centrifuged for 10min at 7000 g. Remaining lipids and trichloric acid were extracted from the supernatant by water saturated diethyl ether. The aqueous phase was lyophilized, taken up in small volumes of water and separated by thin layer chromatography according to SZABOand BURKE (1972), or DIGHE, PAH~JJAand SHA (1969). Assay of cyclic nucleotide phosphodiesterase
10 20 30
60
Determination trations
of
endogenous
cyclic
AMP
concen-
Plexuses were incubated in Krebs-Henseleit solution at 37°C for 5min in the presence or absence of drugs. After homogenization in 6% trichloric acid extraction of lipids and trichloric acid by diethyl ether and subsequent lyophilisation, the samples were taken up in 100 ~1 of 40 mu Tris pH 7.4 and purified on aluminium oxide columns according as described by RAMACHANDRAN (1971). Media were lyophiliied without prior purification. Cyclic AMP in the lyophilized effluent and the medium was determined according to the method of GILMAN(1970). RESULTS
activity
Plexuses were homogenized in 0.04~ imidazole buffer (pH 7.6). According to THOMPSONand APPLEMAN(1971) 100~1 of the homogenate was incubated at 30°C in a solution composed of 20 ~1 0.4~ Tris pH 8.0, 20 ~1 0.05 M MgS04, 20 ~1 labelled and 20 ~1 unlabelled cyclic nucleotide as well as 50 ~1 of snake venom nucleotidase (Sigma Co). After incubation for 10 min the reaction was stopped by boiling for 2 min. The labelled nucleoside was separated from labelled
0
nucleotides by 500~1 of a Dowex 1 x 2 suspension and counted in Instagel. Results were expressed as pmol cyclic, nucleotide hydrolized per mg protein per min. The protein concentration was measured according to LOWRY, ROSIZIROUGH,FARR and RANDALL (1951).
Uptake of [8-3H]-cyclic AMP and [8-3H]-cyclic by choroid plexus and pia mater
GMP
The time course of uptake of radioactivity into isolated rabbit choroid plexus incubated with [3H]-cyclit AMP or [3H]-cyclic GMP is shown in Figure 1. Both nucleotides in 1 pM concentration appeared to accumulate at tissue/medium ratios of 4.0 + 0.8 and 3.2 _+ 1.0 respectively after 10min of incubation. The accumulation of radioactivity proceeded almost
1
120 TIMEOF INCUBAllON lminl
~C8-3H1-cyclic
AMP
:[8-3HI-cyclic
GMP
I 240
Fig. 1. Uptake of radioactivity, expressed as tissue/medium (T/M) ratio, by isolated rabbit choroid plexus and pieces of pia mater incubated with 1.0 pM [8-“HI-cyclic AMP and 1.0PM [8-3H]-cyclic
GMP. Values for choroid plexus are means _+ S.D. (n = 3-Q, values for pia mater tissue are results of single incubations (about 20 mg tissue for each point).
Cyclic AMP in rabbit choroid plexus
3-
137
0 [8-3 HI -cyclic
0
2 E
l
f8-3Hl-cyc~ic
AMP GMP
:
2-
o*
I 10“
I 10S5
r 10“ SlJWRAlE
I 10”
I lo-’
1O”M
CONCENlRATlON
Fig. 2. Uptake of radioactivity by isolated rabbit choroid plexus incubated for 10min with [8-“HI-cyclic AMP and [8-3H]-cyclic GMP as a function of substrate concentration. Tissue uptake is expressed as tissue/medium (T/M) ratio f S.D. (n = 3-12).
Iinearly for 4 hr reaching tissue/medium ratios of 89 + 2.4 and 83 _+ 2.5 respectively. Uptake of radioactivity from [3H]-cyclic AMP and [3H]-cyclic GMP into isolated pieces of rabbit pia mater under identical conditions proved to be linear ffor at least 2 hr (Fig. 1). The tissue/medium ratios were about the same for both nucleotides and 50% of those obtained in choroid plexus. An increase of the concentration of both labelled nucleotides in the medium to 10 mu caused a nearly exponential decline of the 10 min tissue/medium ratios (Fig. 2). No further
decline however was seen between 10 and 50 mu, suggesting that the process of accumulation involved a saturable as well as non-saturable, e.g. diffusional, component. Influence of metabolic inhibition, ouabain and omission of sodium on the uptake of [8-3H]-cyclic AMP The data presented in Table 1 show that the uptake of labelled cyclic AMP was reduced by lowering the incubation temperature and by anoxia. When the 95% oxygen atmosphere was replaced by nitrogen, the
Table 1. Uptake of [8-3H]-cyclic AMP into the rabbit choroid plexus in vitro
Condition 95% N2 + 5% CO2 Temperature 27°C Dinitrophenol (2 mM) Ouabain (0.1mM) Sodium free medium Cyclic GMP (5 mhr) S-AMP (5 mM) Benzyl-penicillin (5 IIIM) 5-HIAA (5 M) p-Aminohippuric acid (5 mM) Probenecid (5 mt.r) L-Proline 5 mM L-Histidine (5 mu) Choline-Cl (5 mM)
Percentage of individual control
T/M + SD. 2.65 f: 0.60 (n = 2.94 f 0.58 (n = 1.95 +_0.18 (n = 1.88 + 0.30 (n = 4.78 * 0.80 (n = 1.11 It 0.25 (n = 1.68 f 0.50 (n = 2.39 f 0.87 (n = 3.99 + 0.20 (n = 3.44 + 0.79 (n = 3.35 f 0.25 (n = 7.32 * 1.66 (n = 5.23 k 0.76 (n = 6.00 f 0.97 (n =
4) 4) 4) 4) 5) 4)
4) 4) 4) 4) 4) 4) 4) 4)
50**
55** 27’** 55’ 71* 19*** 2a*** 38** 63* 55* 43*** 100 80 92
Effects of metabolic inhibition and of various drugs ou the tissue/medium (T/M) ratios for 1 PM[‘HI-cyclic AMP were determined after 20 min incubation. The results were matched with individual controls (95% O2 + 5% C02; 370C; Na+ 141mhr). *P < 0.0125; **P < 0.0025; *** P < 0.0005.
N.P. 16/2--2
R. HAMMERS,P. CLARENBACH. T. LINDL and H. CRAMER
138
3), whereas the neutral and basic substances tested were without significant effect (Table 1). Metabolism of [8-3H]-cyclic AMP in the tissue and in the incubation medium. Influence of theophylline In initial experiments choroid plexuses exposed to labelled cyclic AMP for 60min
were
under standard conditions (see Fig. l), washed three times and again incubated in nucleotide-free medium for various intervals. During this second incubation, tissue radioactivity declined exponentially by about 40% within lOmin, but only by an additional 5% within the next 2 hr. This indicated that after incubation of 60min more than half of the radioactivity accumulated by the choroid plexus was present in a non-diffusable form. Thin layer chromatography of the media showed Fig. 3. Uptake of radioactivity by isolated rabbit choroid a slight degradation of the labelled cyclic nucleotide plexus incubated with l.OpM [8-3H]-cyclic AMP in the within the medium or a moderate release of labelled presence of various concentrations of probenecid. Results metabolites from the choroid plexus (Table 2). of 20min incubations are expressed as mean tissue/ Main degradation products (not listed in Table 2) medium (T/M) ratios f S.D. (n = 4). were S-AMP and adenosine, and after longer periods inosine and hypoxa$hine also. In contrast, identification of intracellular radioacti20min tissue/min ratio was decreased by 50%. Similarly, 2m~ dinitrophenol markedly reduced the vity showed that only a small percentage could be accounted for by cyclic AMP itself; values obtained uptake of labelled cyclic AMP. Addition of ouabain to the medium (0.1 mM) in repeated experiments ranged between 6 and 15% resulted in a 20min tissue/medium ratio of 1.88, for the 10 min interval, and declined to less’than 1% after 4 hr. The results of a representative experiment representing 45% inhibition. The omission of sodium are shown in Table 2. Whereas 46.6 and 59.4% of led to a significant inhibition of about 30%. the intracellular radioactivity could be identified as Competitive inhibition by dzfirent compounds of the the sum of ATP and ADP after 10 and 60 min incubauptake of [%‘H]-cyclic AMP tion respectively, only 8.5 and 2.3% respectively were identified as cyclic AMP. The rest of the radioactivity Incubation with the structurally related compounds was distributed among other cyclic AMP metabolites cyclic GMP and S-AMP at high concentrations (5m~) inhibited the uptake of labelled cyclic AMP such as S-AMP, adenosine, hypoxanthine and inby 81 and 72% respectively (Table 1). Since both nu- osine. Adenine was found in trace amounts only. On cleotides might interfere with the intracellular meta- the other hand, a small but increasing portion of bolism of cyclic AMP, other substances known to be radioactivity was associated with the acid insoluble actively transported out of CSF and/or to be concen- fraction of the tissue homogenates (1.8x, 5.5% and trated by choroid plexus in vitro, were tested. Signifi- 9.6% of the total radioactivity after 10, 60 and cant inhibition of [3H]-cyclic AMP uptake was 240 min incubation respectively), indicating incorporation into macro-molecular compounds such as DNA observed with the organic acids benzyl-penicillin, 5-HIAA, p-aminohippuric acid and probenecid (Fig. or RNA.
Table 2. The accumulation of radioactivity in the rabbit choroid plexus during incubation with [8-3H]-cyclic AMP (l.Op~), the resulting distribution of cyclic AMP and ATP/ADP within the incubation medium and the plexus tissue, and the effect of 2rn~ theophylline on both (values in parenthesis) Time of incubation (min)
T/M + S.D.
0
10 60
Medium % cyclic AMP %ATP + ADP
Choroid plexus % cyclic AMP %ATP + ADP
3.1 * 0.2 (1.0 17.0*+ 0.1) 2.2
93.1 (93.2) 89.9 (?9$)
0.0 (0.0) 0.2 (i.i)
(2:::)
(4.5 + 0.6)
(82:4)
(0:2)
(Z,
Thin layer chromatographic and BURKE,1972).
46.6 (26.6) 59.4 (58.4)
analysis of tissue extracts and media was performed on polyethyleneimine-cellulose
(SZABO
Cyclic AMP in rabbit choroid plexus
139
As shown in Table 2, the uptake of labelled nucleotides also depended on the activity of cyclic nucleotide phosphodiesterases which could be shown to be present in crude homogenates of choroid plexus with a specific activity of 50 pmol/mg protein per min for both cyclic AMP and cyclic GMP. The inhibition of phosphodiesterase activity by 2rn~ theophylline decreased the tissue/medium ratio of labelled cyclic AMP by about two thirds, while tissue [3H]-cyclic AMP was three times higher than under control conditions.
cranial and spinal arachnoid villi, and by exchange of CSF constituents with plasma via transport processes at choroidal and extrachoroidal sites (Cswc, 1971). We were interested in the mechanisms by which cyclic AMP, alter being released into CSF from cerebral tissues, is eliminated from the CSF compartment. Earlier observations in the intact animal (CRAMERand LINDL, 1972) and in man (CRAMERet al., 1972a, b), suggested that cyclic AMP is cleared from CSF by a probenecid-sensitive transport mechanism, possibly located in the choroid plexus. The results presented show that rabbit choroid plexus Occurence and formation of endogenous cyclic AMP and pia mater are capable of removing both cyclic in the isolated choroid plexus AMP and cyclic GMP from the bathing fluid in vitro. The uptake of cyclic AMP appeared to be saturable At zero time and increasingly during incubations, in part, to depend on temperature and oxidative labelled metabolites of [3H]-cyclic AMP, mainly C3H]-SAMP and C3H]-adenosine, were shown to be metabolism, to be coupled to the activity of a K’Na+-dependent ATPase and reduced by dinitropresent in the medium. Reuptake of these compounds phenol. Thus, the uptake satisfies a number of criteria can be assumed to contribute to the obvious accumulation of radioactivity in the choroid plexus tissue. generally accepted for active membrane transport Moreover, it is not excluded that uptake per se pre- (CSERR,1971). Additional findings suggested that cyclic nucleodominantly or even exclusively occurs after prior degradation of the cyclic nucleotide, i.e. hydrolysis or tides are transported by the same carrier as other dephosphorylation in the medium and subsequent in- organic weak anions since the transport was inhibited by the organic acids benzyl-penicillin, 5-HIAA and ward-transport of the metabolites. Thus the intracellular labelled cyclic AMP would be the product of p-aminohippuric acid, whereas no interference was seen with neutral or basic amino acids. Furthermore resynthesis. a dose-dependent inhibition of cyclic AMP uptake A mechanism like this which has been demonstrated in cell preparations of thyroid tissue (SZABO was observed with probenecid, which is supposed to and BURKE, 1972) would require an extracellular or competitively inhibit the weak anion carrier in the kidney (COULSON,BOWMANand ROCH-RAMEL,1974) at least cell membrane associated phosphodiesterase and in the choroid plexus of the cerebral ventricles and a cyclic AMP generating system in the choroid (ANDERSSON and Roes, 1972), and which increases plexus cells. To evaluate the cyclic AMP synthesizing capacity of the plexus, the cyclic AMP content was CSF cyclic AMP levels in vivo in man (CRAMERet determined under several conditions using the al., 1972a, b) and in animals (CRAMERand LINDL, method of GILMAN(1970). The mean value of cyclic 1972). However, in contrast to the cyclic nucleotides AMP in rabbit choroid plexus incubated in drug free medium for 5 min at 37°C was 59.9 + 10.5 pmol/mg protein. Small but measurable amounts of cyclic AMP also were present in the incubation medium (Fig. 4). Theophylline (2m~) was unable to increase this ‘basal’ cyclic AMP concentration. Noradrenaline, which is found in the choroid plexus presumably assatiated with sympathetic nerve endings, was unable to increase cyclic AMP levels in the isolated choroid plexus when tested at concentrations up to 0.5m~ (Fig. 4). Histamine, a potent stimulator of central (KAKIUCHI and RALL, 1968) as well as peripheral (LINDL and CRAMER,1974) nervous tissue adenylate cyclase induced a nearly fourfold increase in the concentration of cyclic AMP after Smin incubation at 0.5 mM concentration. Prostaglandin El had a moderate but significant effect on cyclic AMP levels at O.ClOlmM concentrations as shown in a representative t%E,.IN IN WA-IN NI.lN experiment (Fig. 4). DISCUSSIOh Jle chemical constitution of CSF is regulated both by bulk absorption of fluid into venous blood at
Fig. 4. Levels of cyclic AMP in isolated rabbit choroid
plexus after incubations of 5 min in the presence of tissue ‘hormones; 0.5 mM noradrenaline (NA), 0.5 mM histamine (HI), 0.001 mM PGE,, 2 nw theophylline (TH). Hatched bars represent cyclic AMP in the incubation medium.
140
R. HAMMERS, P. CLARENBACH, T. LINDLand H. CRAMER
none of the metabolites or drugs previously shown to be taken up by the choroid plexus in vitro ever reached tissue/medium values as high as 80, which suggested involvement of other factors than simple transport in the uptake process for cyclic nucleotides. Renal clearance of cyclic AMP from plasma recently has been found to involve degradation of the nucleotide in the renal cortex, while most of the cyclic AMP excreted with urine, where it occurs in concentrations about 100 times higher than in the plgsma, appeared to originate in the kidneys themselves (COULSONet al., 1974). Therefore it was important to investigate if similar mechanisms, i.e. degradation and perhaps resynthesis, could account for the very high uptake of radioactivity by choroid plexus in vitro, and possibly the rapid clearance of cyclic AMP from CSF in uioo. Thin layer chromatography of plexus extracts revealed that both in the medium and in the plexus radioactivity was associated to a large extent with cyclic AMP metabolites after short periods of incubation. The potency of choroid plexus to hydrolyze cyclic nucleotides was further substantiated by measurements of cyclic nucleotide phosphodiesterase activity in choroid plexus homogenates, which revealed a high specific activity of the enzyme for both cyclic AMP and cyclic GMP. Thus, at least a number of the apparent criteria of active transport, namely saturability and inhibition by metabolic inhibitors, can be accounted for by the metabolism of the nucleotide in the plexus. It was found that cyclic AMP is rapidly metabolized to non-diffusable metabolites such as ATP and to diffusable metabolites such as adenosine, and that rapid labelling of endogenous adenine nucleotide pools contributes to the extremely high uptake of radioactivity from 8-C3H]cyclic AMP and to the fast changes in the distribution of labelled adenine nucleotide in the plexus tissue. In addition, a substantial part of the radioactivity was incorporated into acid insoluble components, presumably DNA and RNA. SZABOand BURKE(1972) suggested that in suspended cells of the thyroid gland uptake of cyclic AMP metabolites and subsequent resynthesis of cyclic AMP accounts for the appearance of tritium labelled cyclic AMP in the tissue. In our experiments, the remarkably high concentration of cyclic AMP may suggest that in the choroid plexus cyclic AMP is, at least in part, synthesized in situ and not taken up from CSF. The virtual absence of cyclic AMP phosphodiesterase activity in normal CSF, however, makes it unlikely that cyclic AMP is degraded prior to uptake as suggested by SZABOand BURKE(1972) for thyroid cell-suspensions. The findings of both high endogenous cyclic AMP levels and a hormone-sensitive cyclic AMP generating system in the rabbit choroid plexus suggests that cyclic AMP may have a role of its own in the metabolism and function of this tissue. Noradrenaline, which occurs in considerable amounts in the choroid plexus (EDVINSSON, OWMAN, ROSENGRENand WEST, 1972) was unable to increase cyclic AMP levels in this tis-
sue, whereas histamine which stimulates cyclic AMP synthesis in the brain (KAKIUCHI and RALL, 1968), capillary-enriched fractions of brain tissue (Joo, RAKONCZAYand WOLLMANN, 1975) and in peripheral sympathetic ganglia (LINDL and CRAMER,1974) induced a rapid accumulation of cyclic AMP in the plexus. Prostaglandin E,, which increases cyclic AMP in rat brain in viva (WELLMANNand SCHWABE,1973) was also able to increase plexus cyclic AMP levels. Together, these findings lead us to propose two hypotheses concerning the fate and functions of cyclic AMP in the choroid plexus. First, inactivation by hydrolysis through phosphodiesterase activity in the choroid plexus and possibly other structures lining the CSF space may be an important part of the mechanisms of cyclic nucleotide clearance from the CSF in duo. This hypothesis is also supported by the observation that theophylhne, which in this study has been shown to reduce uptake and to inhibit hydrolysis of cyclic AMP, induces a marked accumulation of cyclic AMP in rat CSF in uiuo (KIESSLING,LMDL and CRAMER,1976), without significant increases in brain cyclic AMP (CRAMER,unpublished observation). The second hypothesis derives from our observation of a cyclic AMP generating system which is stimulated by histamine and prostaglandin E1 and proposes an as yet unknown role of cyclic AMP in choroid plexus function. Since cyclic AMP appears to be implicated in permeability changes at brain capillaries (Jo0 et al., 1975), cyclic AMP may well have a role in the regulation of transport processes at blood-brain and blood-CSF barriers. The precise location of the cyclic AMP generating system in the epithelium or in the stroma and capillaries of the choroid plexus will be crucial in the determination of cyclic AMP functions in this tissue. REFERENCES ANDERSSON, H. and Roos, B.-E. (1972).S-Hydroxyindoleacetic acid and homovanillic acid in cerebrospinal fluid and brain of different rabbit breeds after treatment with probenecid. J. Pharm. Pharmac. 24: 165-166. BERING,E. A., JR. (1955). Choroid plexus and arterial pulsation of cerebrospinal fluid. Demonstration of the choroid plexus as a cerebrospinal fluid pump. Archs Neural. Psychiat. 73: 165-172. COULSON,R., BOWMAN,R. H. and ROCH-RAMZL., F. (1974). The effects of nephrectomy and probenecid on in uiuo clearance of adenosine-3’,5’-monophosphate from rat plasma. Lije Sci. 15:871-886. CRAMER,H., GOODWIN, F. K., POST, R. M. and BUNNEY, E. E. (1972a). Effects of probenecid and exercise on cerebrospinal fluid cyclic AMP in affective illness. Lancer 1: 13461347. CRAMER,H. and LINDL, T. (1972). Probenecid inhibits efflux of adenosine 3’,5’-monophosphate (CAMP) from cerebrospinal fluid in the rat. Psychopharmacologia 26: Suppl. 49. CRAMER,H. and LINDL,T. (1974). Release of cyclic AMP from rat superior cervical ganglia after stimulation of synthesis in vitro. Nature, Lord. 249: 38G382. CRAMER,H, NG, L. K. Y. and CHASE,T. N. (1972b). Cyclic AMP: probenecid-induced rise in human cerebrospinal fluid. J. Neurochem. 19: 1601-1602.
Cyclic AMP in rabbit choroid plexus CSERR,H. (1971). Physiology of the choroid plexus. Physiol. Rev. 51: 273-311. CSERR,H. and VANDYKE,D. H. (1971). S-Hydroxyindoleacetic acid accumulation by isolated choroid plexus. Am. J. Physiol. 224k 718-723. DIGHE.P. K.. PAHUJA.D. N. and SHA. D. H. (1969). Seuaration of cyclic 3’,5’-AMP from other adenine nucleotihes. J. Chromat. 40: 449-452. EDVINSSON,L., OWMAN, CH., ROSENGREN, E. and WEST, K. A. (1972). Concentration of noradrenaline in pial vessels, choroid plexus, and iris during two weeks after sympathetic ganghonectomy or decentralization. Acta physiol. stand. 85: 201-206. FORN, J. (1972). Active transport of 5hydroxyindoleacetic acid by the choroid plexus in vitro. Blockade by probenecid and metabolic inhibitors. Biochem. Pharmac. 21: 619424. GILMAN,A. G. (1970). A protein binding assay for adenosine 3’,5’-cyclic monophosphate. Proc. Natn. Acad. Sci. U.S.A. 67: 305-312. GULDBERG,H. C., ASHCROFT,G. and CRAWFORD,T. B. B. (1966). Concentrations of 5hydroxyindoleacetic acid and homovanilhc acid in the cerebrospinal fluid of the dog before and after treatment with probenecid. Life Sci. 5: 1571-1575. Joe, F., RAKONCZAY, Z. and WOLLMANN, M. (1975). CAMPmediated regulation of the permeability in the brain capillaries. Experlentia 31: 582-583. KAKIUCHI,S. and RALL, T. W. (1968) The influence of chemical agents on the accumulation of adenosine 3’,5’-phosphate in shces of rabbit cerebellum. Molec. Ph&mac.*rl: 367-378. KIESSLING. M.. LINDL.T.. CRAMER.H. (1975). Cvclic adenosine monophosphate ;n cerebrospmal &id. Effects of theophylhne. L-DOPA and a dopamine receptor stimulant in rats. Arch. Psychiat. Nero Krankh. 220: 325-333. LINDL, T. and CRAMER,H. (1974). Formation, accumu-
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lation and release of adenosine 3’,5’-monophosphate induced by histamine in the superior cervical ganglion of the rat in vitro. Biochim. biophys. Acta 343: 182-191. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, J. (1951). Protein measurement with the fohn DhenoJ reagent. J. biol. Chem. 193: 265-275. N~FF, N. H., TOZER,T. N. and BRODIE,B. B. (1967). Application of steady-state kinetics to studies of the transfer of 5-hydroxy-indoleacetic acid from brain to plasma. J. Pharmac. exp. Ther. 158: 214-218. PAPPENHEIMER, J. R., HEISEY,S. R. and JORDAN,E. F. (1961). Active transport of Diodrast and phenolsulfonphtalein from cerebrospinal fluid to blood. Am. J. Physiol. 2ooz l-10. RAMACHANDRAN, J. (1971). A new simple method for separation of adenosine 3’,5’-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Ana2yt. Biochem. 43: 227-239. ROEII~ON,G. A., COPPEN, A. J., WHYBROW,P. C. and FLANGE, A. J. (1970). Cyclic AMP in affective disorders. Lancet 2: 1028. ROUGEMENT, J., DE, AMES,A., NESBETT, F. B. and HOFMANN, H. F. (1960). Fluid formed by choroid plexus. A technique for its collection and a comparison of its electrolyte composition with serum and cistemal fluids. J. Neurophysiol. U: 485-495. SZALIO,M. and BURKE,G. (1972). Uptake and metabolism of 3’,5’-cyclic adenosine monophosphate and N6. 02’dibutyryl 3’,5’-cyclic adenosine monophosphate in isolated bovine thyroid cells. Biochim. biophys. Acta 264: 289-299. THOMPSON, W. J. and APPLEMAN,M. M. (1971). Multiple cyclic nucleotide phosphodiesterase activities from rat brain, Biochemistry 16 31 l-317. WELLMANN. . ’ W. and SCHWABE.U. (1973). Effects of orostaglandms Et, E, and Fza on cychc AMP levels in brain in vivo. Brain Rex 59: 371-378.
1977, 16, 135-141.
Pergamon
Press.
Printed
in Gt. Britain.
UPTAKE AND METABOLISM OF CYCLIC AMP IN RABBIT CHOROID PLEXUS IN VITRO* R. HAMMERS,P. CLARJZNBACH, T. LINDL~ and H. CRAMER Department of Neurology, University of Freiburg, Freiburg, FRG (Accepted 22 June 1976) Summary-The uptake and fate of labelled cyclic nucleotides in isolated rabbit choroid plexus were studied in vitro. Radioactivity accumulated almost linearily for 240 min, reaching tissue/medium ratios of 89 f 2.4 for cyclic AMP and 83 f 2.5 for cyclic GMP. The uptake process was saturable at substrate concentrations ranging from l.OpM to 10mM. The uptake of [3H]-cyclic AMP was significantly inhibited by lowering of temperature, anoxia, ouabain and omission of sodium as well as by cyclic GMP, S-AMP, probenecid and several other organic acids but not by neutral or basic compounds. Thin layer chromatographic identification of the intracellular radioactivity revealed that after 10 min incubation less than 10% was attributable to cyclic AMP itself, whereas about 45% was associated with ATP + ADP and 2% with the acid insoluble fraction of the choroid plexus. Inhibition of phosphodiesterase activity by theophylline increased the intracellular cyclic AMP fraction of total radioactivity accumulated, but decreased the tissue/medium ratio by about two thirds. The finding of a cyclic AMP generating system sensitive to histamine and prostaglandin E,, but not to noradrenaline, in the choroid plexus suggests a functional role of cyclic AMP in this tissue.
The choroid plexuses of the ventricular cavities are the major sites of production of ccrebrospinal fluid (CSF). (BERING, 1955; ROUGEMONT, AMES, NESBETT and HOFMANN, 1960). More recently data have accumulated suggesting that the choroid plexuses, as well as other parts of the ventricular lining and the spinal pia mater, have an important function in removing metaholites which have entered the CSF compartment from the brain tissue (CSERR, 1971). For the monoamine metabolites homovanillic acid (HVA) and 5hydroxyindoleacetic acid (5HIAA) as well as for a number of foreign compounds and drugs, the transport from CSF to choroidal venous blood has been studied extensively suggesting the existence of mediated transport with several distinct carriers (P~PPENHEIMER,HEISEYand JORDAN, 1961; CSERR and VAN DYKE, 1971. Another brain metabolite, 3’,5’-cyclit adenosine monophosphate (cyclic AMP), occurs in mammalian CSF in concentrations similar to those of the plasma, (ROBISON, COPPEN, WHYBROW and F’RANGE, 1970; CRAMER, GOODWIN, POST and BUNNEY, 1972a) but concentrations vary independently in both compartments (CRAMER and LINDL, 1972; CRAMER, NG and CHASE, 197217). Similarly to the acidic metabolites of monoamines, cyclic AMP ac-
cumulates in CSF after the administration of probenecid (CRAMER, GOODWIN, POST and BUNNEY, 1972a), a drug which inhibits carrier mediated transport of organic anions in the plexus (GULDBERG,ASHCROFT and CRAWFORD,1966; NEFF, TOZER and BRODIE, 1967; FORN 1972). From isolated intact nervous tissue cyclic AMP is released in considerable amounts in relationship to its intracellular formation (CRAMER and LINDL, 1974), and it can be hypothesized that extracellular extrusion, passage into the CSF and transport out of CSF via the choroid plexus and pia mater might be an additional mechanism of inactivation of this cyclic nucleotide in the central nervous system. In order to test further this hypothesis we studied the uptake and fate of labelled cyclic nucleotides in the isolated choroid plexus of the rabbit in vitro. METHODS Randomly bred male rabbits of 2-3.5 kg body weight were killed by cervical dislocation. After rapid removal of the brain the lateral ventricles were opened, their choroid plexuses excised and transferred into chilled Krebs-Henseleit solution. Pial tissue was stripped from the convexity of the hemispheres and treated similarly.
* Supported by the Deutsche Forschungsgemeinschaft (SFB 70). Work presented in this paper is part of a thesis to be submitted to the Medical Faculty of the University of Preiburg (R.H.). t Resent address: Department of Biology, University of Konstanz, D-775 Konst&z. Reprint requests to: Professor H. Cramer. Department of Neurology, University of Freiburg, D-7800 Ereiburg, FRG. -Key words: adenosine 3$monophosphate, guanosine 3,5-monophosphate, cerebrospinal fluid, choroid plexus, tramport, phosphodiesterase, theophylline, histamine.
Uptake of cyclic nucleotides Each incubation tube contained 2 plexuses in 4ml of Krebs-Henseleit solution aerated with 95% O2 and 5% COz (pH 7.4). Unlahelled cyclic AMP or 3’,5’-cyclit guanosine monophosphate (cyclic GMP) in various concentrations and labelled nucleotide (0.5 &i/ml; specific activities: 27.5 Ci/mmol for cyclic AMP and 15.0Ci/mmol for cyclic GMP, both from Amersham Ltd.) were added.
135
R. HAMMERS,P. CLARENBACH, T. LINDLand H. CRAMER
136
After incubations at 37°C from 10 to 24Omin, the plexuses were removed from the medium, washed, transferred into scintillation vials and dissolved with Soluene TM 100. The hydrolysate was neutralized with concentrated HCl for higher counting efficiency, dissolved in butyl-PBD-toluol scintillation fluid and counted. Radioactivity was also counted in the media. After correction for quenching and tissue/water ratio the results were expressed as tissue/medium ratio. Isolation ofcyclic AMP metabolites by thin layer chromatography
After incubation the plexuses were homogenized in 6% trichloric acid and centrifuged for 10min at 7000 g. Remaining lipids and trichloric acid were extracted from the supernatant by water saturated diethyl ether. The aqueous phase was lyophilized, taken up in small volumes of water and separated by thin layer chromatography according to SZABOand BURKE (1972), or DIGHE, PAH~JJAand SHA (1969). Assay of cyclic nucleotide phosphodiesterase
10 20 30
60
Determination trations
of
endogenous
cyclic
AMP
concen-
Plexuses were incubated in Krebs-Henseleit solution at 37°C for 5min in the presence or absence of drugs. After homogenization in 6% trichloric acid extraction of lipids and trichloric acid by diethyl ether and subsequent lyophilisation, the samples were taken up in 100 ~1 of 40 mu Tris pH 7.4 and purified on aluminium oxide columns according as described by RAMACHANDRAN (1971). Media were lyophiliied without prior purification. Cyclic AMP in the lyophilized effluent and the medium was determined according to the method of GILMAN(1970). RESULTS
activity
Plexuses were homogenized in 0.04~ imidazole buffer (pH 7.6). According to THOMPSONand APPLEMAN(1971) 100~1 of the homogenate was incubated at 30°C in a solution composed of 20 ~1 0.4~ Tris pH 8.0, 20 ~1 0.05 M MgS04, 20 ~1 labelled and 20 ~1 unlabelled cyclic nucleotide as well as 50 ~1 of snake venom nucleotidase (Sigma Co). After incubation for 10 min the reaction was stopped by boiling for 2 min. The labelled nucleoside was separated from labelled
0
nucleotides by 500~1 of a Dowex 1 x 2 suspension and counted in Instagel. Results were expressed as pmol cyclic, nucleotide hydrolized per mg protein per min. The protein concentration was measured according to LOWRY, ROSIZIROUGH,FARR and RANDALL (1951).
Uptake of [8-3H]-cyclic AMP and [8-3H]-cyclic by choroid plexus and pia mater
GMP
The time course of uptake of radioactivity into isolated rabbit choroid plexus incubated with [3H]-cyclit AMP or [3H]-cyclic GMP is shown in Figure 1. Both nucleotides in 1 pM concentration appeared to accumulate at tissue/medium ratios of 4.0 + 0.8 and 3.2 _+ 1.0 respectively after 10min of incubation. The accumulation of radioactivity proceeded almost
1
120 TIMEOF INCUBAllON lminl
~C8-3H1-cyclic
AMP
:[8-3HI-cyclic
GMP
I 240
Fig. 1. Uptake of radioactivity, expressed as tissue/medium (T/M) ratio, by isolated rabbit choroid plexus and pieces of pia mater incubated with 1.0 pM [8-“HI-cyclic AMP and 1.0PM [8-3H]-cyclic
GMP. Values for choroid plexus are means _+ S.D. (n = 3-Q, values for pia mater tissue are results of single incubations (about 20 mg tissue for each point).
Cyclic AMP in rabbit choroid plexus
3-
137
0 [8-3 HI -cyclic
0
2 E
l
f8-3Hl-cyc~ic
AMP GMP
:
2-
o*
I 10“
I 10S5
r 10“ SlJWRAlE
I 10”
I lo-’
1O”M
CONCENlRATlON
Fig. 2. Uptake of radioactivity by isolated rabbit choroid plexus incubated for 10min with [8-“HI-cyclic AMP and [8-3H]-cyclic GMP as a function of substrate concentration. Tissue uptake is expressed as tissue/medium (T/M) ratio f S.D. (n = 3-12).
Iinearly for 4 hr reaching tissue/medium ratios of 89 + 2.4 and 83 _+ 2.5 respectively. Uptake of radioactivity from [3H]-cyclic AMP and [3H]-cyclic GMP into isolated pieces of rabbit pia mater under identical conditions proved to be linear ffor at least 2 hr (Fig. 1). The tissue/medium ratios were about the same for both nucleotides and 50% of those obtained in choroid plexus. An increase of the concentration of both labelled nucleotides in the medium to 10 mu caused a nearly exponential decline of the 10 min tissue/medium ratios (Fig. 2). No further
decline however was seen between 10 and 50 mu, suggesting that the process of accumulation involved a saturable as well as non-saturable, e.g. diffusional, component. Influence of metabolic inhibition, ouabain and omission of sodium on the uptake of [8-3H]-cyclic AMP The data presented in Table 1 show that the uptake of labelled cyclic AMP was reduced by lowering the incubation temperature and by anoxia. When the 95% oxygen atmosphere was replaced by nitrogen, the
Table 1. Uptake of [8-3H]-cyclic AMP into the rabbit choroid plexus in vitro
Condition 95% N2 + 5% CO2 Temperature 27°C Dinitrophenol (2 mM) Ouabain (0.1mM) Sodium free medium Cyclic GMP (5 mhr) S-AMP (5 mM) Benzyl-penicillin (5 IIIM) 5-HIAA (5 M) p-Aminohippuric acid (5 mM) Probenecid (5 mt.r) L-Proline 5 mM L-Histidine (5 mu) Choline-Cl (5 mM)
Percentage of individual control
T/M + SD. 2.65 f: 0.60 (n = 2.94 f 0.58 (n = 1.95 +_0.18 (n = 1.88 + 0.30 (n = 4.78 * 0.80 (n = 1.11 It 0.25 (n = 1.68 f 0.50 (n = 2.39 f 0.87 (n = 3.99 + 0.20 (n = 3.44 + 0.79 (n = 3.35 f 0.25 (n = 7.32 * 1.66 (n = 5.23 k 0.76 (n = 6.00 f 0.97 (n =
4) 4) 4) 4) 5) 4)
4) 4) 4) 4) 4) 4) 4) 4)
50**
55** 27’** 55’ 71* 19*** 2a*** 38** 63* 55* 43*** 100 80 92
Effects of metabolic inhibition and of various drugs ou the tissue/medium (T/M) ratios for 1 PM[‘HI-cyclic AMP were determined after 20 min incubation. The results were matched with individual controls (95% O2 + 5% C02; 370C; Na+ 141mhr). *P < 0.0125; **P < 0.0025; *** P < 0.0005.
N.P. 16/2--2
R. HAMMERS,P. CLARENBACH. T. LINDL and H. CRAMER
138
3), whereas the neutral and basic substances tested were without significant effect (Table 1). Metabolism of [8-3H]-cyclic AMP in the tissue and in the incubation medium. Influence of theophylline In initial experiments choroid plexuses exposed to labelled cyclic AMP for 60min
were
under standard conditions (see Fig. l), washed three times and again incubated in nucleotide-free medium for various intervals. During this second incubation, tissue radioactivity declined exponentially by about 40% within lOmin, but only by an additional 5% within the next 2 hr. This indicated that after incubation of 60min more than half of the radioactivity accumulated by the choroid plexus was present in a non-diffusable form. Thin layer chromatography of the media showed Fig. 3. Uptake of radioactivity by isolated rabbit choroid a slight degradation of the labelled cyclic nucleotide plexus incubated with l.OpM [8-3H]-cyclic AMP in the within the medium or a moderate release of labelled presence of various concentrations of probenecid. Results metabolites from the choroid plexus (Table 2). of 20min incubations are expressed as mean tissue/ Main degradation products (not listed in Table 2) medium (T/M) ratios f S.D. (n = 4). were S-AMP and adenosine, and after longer periods inosine and hypoxa$hine also. In contrast, identification of intracellular radioacti20min tissue/min ratio was decreased by 50%. Similarly, 2m~ dinitrophenol markedly reduced the vity showed that only a small percentage could be accounted for by cyclic AMP itself; values obtained uptake of labelled cyclic AMP. Addition of ouabain to the medium (0.1 mM) in repeated experiments ranged between 6 and 15% resulted in a 20min tissue/medium ratio of 1.88, for the 10 min interval, and declined to less’than 1% after 4 hr. The results of a representative experiment representing 45% inhibition. The omission of sodium are shown in Table 2. Whereas 46.6 and 59.4% of led to a significant inhibition of about 30%. the intracellular radioactivity could be identified as Competitive inhibition by dzfirent compounds of the the sum of ATP and ADP after 10 and 60 min incubauptake of [%‘H]-cyclic AMP tion respectively, only 8.5 and 2.3% respectively were identified as cyclic AMP. The rest of the radioactivity Incubation with the structurally related compounds was distributed among other cyclic AMP metabolites cyclic GMP and S-AMP at high concentrations (5m~) inhibited the uptake of labelled cyclic AMP such as S-AMP, adenosine, hypoxanthine and inby 81 and 72% respectively (Table 1). Since both nu- osine. Adenine was found in trace amounts only. On cleotides might interfere with the intracellular meta- the other hand, a small but increasing portion of bolism of cyclic AMP, other substances known to be radioactivity was associated with the acid insoluble actively transported out of CSF and/or to be concen- fraction of the tissue homogenates (1.8x, 5.5% and trated by choroid plexus in vitro, were tested. Signifi- 9.6% of the total radioactivity after 10, 60 and cant inhibition of [3H]-cyclic AMP uptake was 240 min incubation respectively), indicating incorporation into macro-molecular compounds such as DNA observed with the organic acids benzyl-penicillin, 5-HIAA, p-aminohippuric acid and probenecid (Fig. or RNA.
Table 2. The accumulation of radioactivity in the rabbit choroid plexus during incubation with [8-3H]-cyclic AMP (l.Op~), the resulting distribution of cyclic AMP and ATP/ADP within the incubation medium and the plexus tissue, and the effect of 2rn~ theophylline on both (values in parenthesis) Time of incubation (min)
T/M + S.D.
0
10 60
Medium % cyclic AMP %ATP + ADP
Choroid plexus % cyclic AMP %ATP + ADP
3.1 * 0.2 (1.0 17.0*+ 0.1) 2.2
93.1 (93.2) 89.9 (?9$)
0.0 (0.0) 0.2 (i.i)
(2:::)
(4.5 + 0.6)
(82:4)
(0:2)
(Z,
Thin layer chromatographic and BURKE,1972).
46.6 (26.6) 59.4 (58.4)
analysis of tissue extracts and media was performed on polyethyleneimine-cellulose
(SZABO
Cyclic AMP in rabbit choroid plexus
139
As shown in Table 2, the uptake of labelled nucleotides also depended on the activity of cyclic nucleotide phosphodiesterases which could be shown to be present in crude homogenates of choroid plexus with a specific activity of 50 pmol/mg protein per min for both cyclic AMP and cyclic GMP. The inhibition of phosphodiesterase activity by 2rn~ theophylline decreased the tissue/medium ratio of labelled cyclic AMP by about two thirds, while tissue [3H]-cyclic AMP was three times higher than under control conditions.
cranial and spinal arachnoid villi, and by exchange of CSF constituents with plasma via transport processes at choroidal and extrachoroidal sites (Cswc, 1971). We were interested in the mechanisms by which cyclic AMP, alter being released into CSF from cerebral tissues, is eliminated from the CSF compartment. Earlier observations in the intact animal (CRAMERand LINDL, 1972) and in man (CRAMERet al., 1972a, b), suggested that cyclic AMP is cleared from CSF by a probenecid-sensitive transport mechanism, possibly located in the choroid plexus. The results presented show that rabbit choroid plexus Occurence and formation of endogenous cyclic AMP and pia mater are capable of removing both cyclic in the isolated choroid plexus AMP and cyclic GMP from the bathing fluid in vitro. The uptake of cyclic AMP appeared to be saturable At zero time and increasingly during incubations, in part, to depend on temperature and oxidative labelled metabolites of [3H]-cyclic AMP, mainly C3H]-SAMP and C3H]-adenosine, were shown to be metabolism, to be coupled to the activity of a K’Na+-dependent ATPase and reduced by dinitropresent in the medium. Reuptake of these compounds phenol. Thus, the uptake satisfies a number of criteria can be assumed to contribute to the obvious accumulation of radioactivity in the choroid plexus tissue. generally accepted for active membrane transport Moreover, it is not excluded that uptake per se pre- (CSERR,1971). Additional findings suggested that cyclic nucleodominantly or even exclusively occurs after prior degradation of the cyclic nucleotide, i.e. hydrolysis or tides are transported by the same carrier as other dephosphorylation in the medium and subsequent in- organic weak anions since the transport was inhibited by the organic acids benzyl-penicillin, 5-HIAA and ward-transport of the metabolites. Thus the intracellular labelled cyclic AMP would be the product of p-aminohippuric acid, whereas no interference was seen with neutral or basic amino acids. Furthermore resynthesis. a dose-dependent inhibition of cyclic AMP uptake A mechanism like this which has been demonstrated in cell preparations of thyroid tissue (SZABO was observed with probenecid, which is supposed to and BURKE, 1972) would require an extracellular or competitively inhibit the weak anion carrier in the kidney (COULSON,BOWMANand ROCH-RAMEL,1974) at least cell membrane associated phosphodiesterase and in the choroid plexus of the cerebral ventricles and a cyclic AMP generating system in the choroid (ANDERSSON and Roes, 1972), and which increases plexus cells. To evaluate the cyclic AMP synthesizing capacity of the plexus, the cyclic AMP content was CSF cyclic AMP levels in vivo in man (CRAMERet determined under several conditions using the al., 1972a, b) and in animals (CRAMERand LINDL, method of GILMAN(1970). The mean value of cyclic 1972). However, in contrast to the cyclic nucleotides AMP in rabbit choroid plexus incubated in drug free medium for 5 min at 37°C was 59.9 + 10.5 pmol/mg protein. Small but measurable amounts of cyclic AMP also were present in the incubation medium (Fig. 4). Theophylline (2m~) was unable to increase this ‘basal’ cyclic AMP concentration. Noradrenaline, which is found in the choroid plexus presumably assatiated with sympathetic nerve endings, was unable to increase cyclic AMP levels in the isolated choroid plexus when tested at concentrations up to 0.5m~ (Fig. 4). Histamine, a potent stimulator of central (KAKIUCHI and RALL, 1968) as well as peripheral (LINDL and CRAMER,1974) nervous tissue adenylate cyclase induced a nearly fourfold increase in the concentration of cyclic AMP after Smin incubation at 0.5 mM concentration. Prostaglandin El had a moderate but significant effect on cyclic AMP levels at O.ClOlmM concentrations as shown in a representative t%E,.IN IN WA-IN NI.lN experiment (Fig. 4). DISCUSSIOh Jle chemical constitution of CSF is regulated both by bulk absorption of fluid into venous blood at
Fig. 4. Levels of cyclic AMP in isolated rabbit choroid
plexus after incubations of 5 min in the presence of tissue ‘hormones; 0.5 mM noradrenaline (NA), 0.5 mM histamine (HI), 0.001 mM PGE,, 2 nw theophylline (TH). Hatched bars represent cyclic AMP in the incubation medium.
140
R. HAMMERS, P. CLARENBACH, T. LINDLand H. CRAMER
none of the metabolites or drugs previously shown to be taken up by the choroid plexus in vitro ever reached tissue/medium values as high as 80, which suggested involvement of other factors than simple transport in the uptake process for cyclic nucleotides. Renal clearance of cyclic AMP from plasma recently has been found to involve degradation of the nucleotide in the renal cortex, while most of the cyclic AMP excreted with urine, where it occurs in concentrations about 100 times higher than in the plgsma, appeared to originate in the kidneys themselves (COULSONet al., 1974). Therefore it was important to investigate if similar mechanisms, i.e. degradation and perhaps resynthesis, could account for the very high uptake of radioactivity by choroid plexus in vitro, and possibly the rapid clearance of cyclic AMP from CSF in uioo. Thin layer chromatography of plexus extracts revealed that both in the medium and in the plexus radioactivity was associated to a large extent with cyclic AMP metabolites after short periods of incubation. The potency of choroid plexus to hydrolyze cyclic nucleotides was further substantiated by measurements of cyclic nucleotide phosphodiesterase activity in choroid plexus homogenates, which revealed a high specific activity of the enzyme for both cyclic AMP and cyclic GMP. Thus, at least a number of the apparent criteria of active transport, namely saturability and inhibition by metabolic inhibitors, can be accounted for by the metabolism of the nucleotide in the plexus. It was found that cyclic AMP is rapidly metabolized to non-diffusable metabolites such as ATP and to diffusable metabolites such as adenosine, and that rapid labelling of endogenous adenine nucleotide pools contributes to the extremely high uptake of radioactivity from 8-C3H]cyclic AMP and to the fast changes in the distribution of labelled adenine nucleotide in the plexus tissue. In addition, a substantial part of the radioactivity was incorporated into acid insoluble components, presumably DNA and RNA. SZABOand BURKE(1972) suggested that in suspended cells of the thyroid gland uptake of cyclic AMP metabolites and subsequent resynthesis of cyclic AMP accounts for the appearance of tritium labelled cyclic AMP in the tissue. In our experiments, the remarkably high concentration of cyclic AMP may suggest that in the choroid plexus cyclic AMP is, at least in part, synthesized in situ and not taken up from CSF. The virtual absence of cyclic AMP phosphodiesterase activity in normal CSF, however, makes it unlikely that cyclic AMP is degraded prior to uptake as suggested by SZABOand BURKE(1972) for thyroid cell-suspensions. The findings of both high endogenous cyclic AMP levels and a hormone-sensitive cyclic AMP generating system in the rabbit choroid plexus suggests that cyclic AMP may have a role of its own in the metabolism and function of this tissue. Noradrenaline, which occurs in considerable amounts in the choroid plexus (EDVINSSON, OWMAN, ROSENGRENand WEST, 1972) was unable to increase cyclic AMP levels in this tis-
sue, whereas histamine which stimulates cyclic AMP synthesis in the brain (KAKIUCHI and RALL, 1968), capillary-enriched fractions of brain tissue (Joo, RAKONCZAYand WOLLMANN, 1975) and in peripheral sympathetic ganglia (LINDL and CRAMER,1974) induced a rapid accumulation of cyclic AMP in the plexus. Prostaglandin E,, which increases cyclic AMP in rat brain in viva (WELLMANNand SCHWABE,1973) was also able to increase plexus cyclic AMP levels. Together, these findings lead us to propose two hypotheses concerning the fate and functions of cyclic AMP in the choroid plexus. First, inactivation by hydrolysis through phosphodiesterase activity in the choroid plexus and possibly other structures lining the CSF space may be an important part of the mechanisms of cyclic nucleotide clearance from the CSF in duo. This hypothesis is also supported by the observation that theophylhne, which in this study has been shown to reduce uptake and to inhibit hydrolysis of cyclic AMP, induces a marked accumulation of cyclic AMP in rat CSF in uiuo (KIESSLING,LMDL and CRAMER,1976), without significant increases in brain cyclic AMP (CRAMER,unpublished observation). The second hypothesis derives from our observation of a cyclic AMP generating system which is stimulated by histamine and prostaglandin E1 and proposes an as yet unknown role of cyclic AMP in choroid plexus function. Since cyclic AMP appears to be implicated in permeability changes at brain capillaries (Jo0 et al., 1975), cyclic AMP may well have a role in the regulation of transport processes at blood-brain and blood-CSF barriers. The precise location of the cyclic AMP generating system in the epithelium or in the stroma and capillaries of the choroid plexus will be crucial in the determination of cyclic AMP functions in this tissue. REFERENCES ANDERSSON, H. and Roos, B.-E. (1972).S-Hydroxyindoleacetic acid and homovanillic acid in cerebrospinal fluid and brain of different rabbit breeds after treatment with probenecid. J. Pharm. Pharmac. 24: 165-166. BERING,E. A., JR. (1955). Choroid plexus and arterial pulsation of cerebrospinal fluid. Demonstration of the choroid plexus as a cerebrospinal fluid pump. Archs Neural. Psychiat. 73: 165-172. COULSON,R., BOWMAN,R. H. and ROCH-RAMZL., F. (1974). The effects of nephrectomy and probenecid on in uiuo clearance of adenosine-3’,5’-monophosphate from rat plasma. Lije Sci. 15:871-886. CRAMER,H., GOODWIN, F. K., POST, R. M. and BUNNEY, E. E. (1972a). Effects of probenecid and exercise on cerebrospinal fluid cyclic AMP in affective illness. Lancer 1: 13461347. CRAMER,H. and LINDL, T. (1972). Probenecid inhibits efflux of adenosine 3’,5’-monophosphate (CAMP) from cerebrospinal fluid in the rat. Psychopharmacologia 26: Suppl. 49. CRAMER,H. and LINDL,T. (1974). Release of cyclic AMP from rat superior cervical ganglia after stimulation of synthesis in vitro. Nature, Lord. 249: 38G382. CRAMER,H, NG, L. K. Y. and CHASE,T. N. (1972b). Cyclic AMP: probenecid-induced rise in human cerebrospinal fluid. J. Neurochem. 19: 1601-1602.
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lation and release of adenosine 3’,5’-monophosphate induced by histamine in the superior cervical ganglion of the rat in vitro. Biochim. biophys. Acta 343: 182-191. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, J. (1951). Protein measurement with the fohn DhenoJ reagent. J. biol. Chem. 193: 265-275. N~FF, N. H., TOZER,T. N. and BRODIE,B. B. (1967). Application of steady-state kinetics to studies of the transfer of 5-hydroxy-indoleacetic acid from brain to plasma. J. Pharmac. exp. Ther. 158: 214-218. PAPPENHEIMER, J. R., HEISEY,S. R. and JORDAN,E. F. (1961). Active transport of Diodrast and phenolsulfonphtalein from cerebrospinal fluid to blood. Am. J. Physiol. 2ooz l-10. RAMACHANDRAN, J. (1971). A new simple method for separation of adenosine 3’,5’-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Ana2yt. Biochem. 43: 227-239. ROEII~ON,G. A., COPPEN, A. J., WHYBROW,P. C. and FLANGE, A. J. (1970). Cyclic AMP in affective disorders. Lancet 2: 1028. ROUGEMENT, J., DE, AMES,A., NESBETT, F. B. and HOFMANN, H. F. (1960). Fluid formed by choroid plexus. A technique for its collection and a comparison of its electrolyte composition with serum and cistemal fluids. J. Neurophysiol. U: 485-495. SZALIO,M. and BURKE,G. (1972). Uptake and metabolism of 3’,5’-cyclic adenosine monophosphate and N6. 02’dibutyryl 3’,5’-cyclic adenosine monophosphate in isolated bovine thyroid cells. Biochim. biophys. Acta 264: 289-299. THOMPSON, W. J. and APPLEMAN,M. M. (1971). Multiple cyclic nucleotide phosphodiesterase activities from rat brain, Biochemistry 16 31 l-317. WELLMANN. . ’ W. and SCHWABE.U. (1973). Effects of orostaglandms Et, E, and Fza on cychc AMP levels in brain in vivo. Brain Rex 59: 371-378.