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Stimulation of cyclic adenosine 3′,5′-monophosphate formation in rabbit choroid plexus by β-receptor agonists and vasoactive intestinal polypeptide
Neuroscience Letters, 54 (1985) 153-157 Elsevier Scientific Publishers Ireland Ltd.
153
NSL 03153
STIMULATION OF CYCLIC ADENOSINE 3' ,5'·MONOPHOSPHATE FORMATION IN RABBIT CHOROID PLEXUS BY .a-RECEPTOR AGONISTS AND VASOACTIVE INTESTINAL POLYPEPTIDE
MARIA LlNDVALL*, A.SA GUSTAFSON, PAVO HEDNER and CHRISTER OWMAN
Departments of Histology and Internal Medicine, University of Lund, Lund (Sweden) (Received June 27th, 1984; Revised version received November 23rd, 1984; Accepted November 26th, 1984)
Key words: choroid plexus - cyclic adenosine 3' ,5 '-monophosphate - {j-receptor agonists - vasoactive intestinal polypeptide
The formation of cyclic adenosine 3',5' -monophosphate (cAMP) was determined in blood-free choroid plexus homogenates from all ventricles of rabbit using a competitive protein binding technique. Previous stimulation of the intact plexus tissue in vitro with sympathomimetic drugs or vasoactive intestinal polypeptide (VIP) leads to increased local synthesis of cAMP. Tests with selective (3-receptor agonists and antagonists suggested that {jt-receptors predominate, which is consistent with studies of sympathomimetic effects on cerebrospinal fluid production in vivo.
The choroid plexus is innervated by adrenergic nerves [5, 11] originating in the superior cervical sympathetic ganglia [6, 11]. The nerve terminals are associated not only with the blood vessels but also with the secretory epithelium of the plexus [4]. Stimulation of the choroidal sympathetic nerves markedly inhibits the formation of cerebrospinal fluid (CSF), whereas the production rate is enhanced following denervation [13]. Pharmacological studies have suggested that the sympathetically mediated inhibition involves both .a-adrenergic receptors on the epithelial cells and ce-adrenoceptors in the vascular bed of the choroid plexus [14]. Studies of transport functions both in vitro [15, 16] and in vivo [20] have confirmed the direct functional influence of the sympathetic system on the secretory epithelium. It has recently been found by immunohistochemistry that the choroid plexus is also innervated by nerve fibers containing vasoactive intestinal polypeptide (VIP) [12]. Adrenergic receptors, particularly of the .a-type, are frequently associated with membrane-bound adenylate cyclase which, following activation, leads to the intracellular synthesis of cyclic adenosine 3' ,5' -monophosphate, referred to as cyclic AMP (cAMP) (cf. ref. 18). VIP also stimulates cAMP formation in several tissues *Author for correspondence at: Department of Histology, Biskopsgatan 5, S-223 62 Lund, Sweden.
154
[1, 2, 7, 10]. The present study on rabbits was undertaken to elucidate whether sympathomimetic stimulation or VIP leads to increased local synthesis of cAMP as a further means to define the functional role of certain adrenergic and peptidergic mechanisms in the choroid plexus. The experiments were performed in 23 randomly pigmented rabbits of either sex weighing 2.5-3.5 kg. They were fed with standard pellet food and tap water ad libitum. The animals were killed by perfusion with 0.9% saline through the left ventricle of the heart under sodium pentobarbital (Mebumal; ACO, Sweden) anesthesia (20 rug/kg i.v.), The amount of saline used in order to obtain completely blood-free plexus tissue was 100 ml. Choroid plexus tissue from each half of the brain including all ventricles was immediately dissected out. Each sample thus contained the plexus from the lateral ventricle and half of the plexus tissue from the third and fourth ventricles. The two batches of material from each animal were put in separate tubes containing 1 ml Krebs-Ringer bicarbonate buffer solution supplied with 10 mM glucose for preincubation during 30 min. Susequent incubation during 10 min was carried out in fresh medium provided with 1 mM theophylline (Sigma). Both incubation steps were performed at 37°C during aeration with a mixture of 9511,70 02-5010 CO 2, After finishing incubation, the tissue was transferred to ice-cold 10% trichloroacetic acid (TCA) for homogenization and the precipitated material was removed by centrifugation at 2500 g in 4°C for 30 min. As a technical control, the supernatant was supplied with 5000 cpm [3H]cAMP (2,8-[3H]adenosine 3',5 -cyclic phosphate, ammonium salt, 30-50 Cilmmol; New England Nuclear) for later estimation of recovery of endogenous cAMP, and put on a Dowex SOW column with 1.5 ml bed volume. cAMP was eluted with distilled water, lyophilized to dryness and resuspended in 500 Itl distilled water for its determination by a modified Gilman [8] method developed by Tovey et al. [23] using charcoal for separation of bound from free cAMP. Recovery was determined by counting a 50 Itl aliquot of the resuspended material. Tissue protein was determined in each tissue sample by the colorimetric method of Lowry et al. [17]. Differences between the mean values of cAMP concentrations in the plexus tissue (expressed as pmol cAMP Img tissue protein) in the different experimental groups were evaluated statistically with Student's t-test. The mean basal level of cAMP in the rabbit choroid plexus after a 10 min incubation period was 48 ± 4 (S.E.M.) pmol/mg protein. The results of the drug effects are summarized in Fig. 1. The (12-adrenoceptor selective agonist, terbutaline (Bricanyl; Draco, Sweden), caused a dose-dependent increase in the cAMP formation. A more pronounced effect was obtained in the presence of the t31-selective agonist H80/62 (Hassle, Sweden). Blockade of the (11-type receptors with practolol (Eraldine; ICI) markedly reduced the effect of H80/62, whereas practolol in combination with terbutaline instead showed a tendency to increased cAMP formation. In the presence of 5 j.tg/ml highly purified porcine VIP (generous gift from Professor Viktor Mutt, Dept. Biochem., Karolinska Institute, Stockholm, Sweden) the
155
cAMP formation was enhanced to the same degree as achieved with 10- 6 M terbutaline in the incubation medium. The choroid plexus is equipped with a cAMP-generating system, suggesting a physiological role of cAMP in the function of this structure [9, 22]. Initial studies failed to activate this system in the rabbit plexus by sympathomimetic stimulation [9, 21], although isoproterenol and epinephrine, as well as norepinephrine, were all able to influence the cAMP content in the rat's choroid plexus. Later, isoproterenol was found to produce an increase in cAMP also in the rabbit plexus tissue [3, 18]. On the basis of the potency rate for the efficiency of various sympathomimetic compounds to stimulate the formation of cAMP in combination with several more or less selective antagonists, Nathanson [19] concluded that the {J-receptor mediating the effect in the feline choroid plexus was of the f32-type. The present findings on rabbit, on the other hand, rather suggest that {JI-receptors predominate. This is consistent with observations on the CSF production rate in the rabbit in vivo,
Treatment
o
cAMP (pmoles/mg protein)
I
Untreated control Terbutaline (l0·6Ml Terbutaline 110"M) 6M)
Terbutaline 110· + practolol (lO"M)
___
50 I
100
150
I
I
---J~ (13) (4)** (5)***
~(4)***
H 80/62 (10- 6M)
(5)***
H 80/62 OO·'M)
H 80/62 (1 0-6M ) +
practolol (lO"M)
(6)***
~(4)***
VIP (SI-lg/ml) Fig. 1. Levels of cAMP determined by protein binding in homogenized rabbit choroid plexus after previous incubations of intact plexus tissue for 10 min in the presence of different sympathomimetic agonists (terbutaline, H80/62), antagonist (practolol) and vasoactive intestinal polypeptide (VIP). Differences from mean control values (± S.E.M.) according to Student's I-test: **0.001< P< 0.01, **"P< 0.001. Number of determinations within parentheses. Controls, unfilled bar; agonists, filled bars; antagonist, hatched bars.
156
where the strong sympathetic inhibition [13J seems to involve {1-receptors in the plexus epithelium of mainly the (31-type [14J. VIP has previously been shown to stimulate cAMP formation in several tissues [1,2, 7, 10J. The formation of cAMP in the rabbit choroid plexus is also stimulated by VIP. Immunohistochemical studies have shown VIP-containing nerve fibers to be present in the structure, particularly around plexus blood vessels, which dilate in response to VIP in vitro [12J. However, considerable species differences were found . In the choroid plexus from pig, a moderate number of VIP-containing nerves was found not only with a perivascular distribution, but also in close association with the plexus epithelium [12J. In contrast, VIP nerves were few in rabbit choroid plexuses [12]. It is thus not unlikely that the VIP-induced stimulation of cAMP formation reflects a functional role of VIP in the choroid plexus, where the putative receptors mediating the peptidergic effect are not necessarily related to the presence of VIP-ergic nerve fibers in the rabbit. Supported by the Swedish Medical Research Council (Grant 04X-732) and by AB Draco, Lund, Sweden.
1 Ahren, B., Alumets, J ., Ericsson, M., Fahrenkrug, L. , HAkanson, R., Hedner, P., Loren, 1., Melander A. , Rerup, C. and Sundler , F., VIP occurs in intrathyroidal nerves and stimulates thyroid hormone secretion , Nature (Lond.), 287 (1980) 343-345. 2 Bataille, D., Freychet, P. and Rosselin, G., Interactions of glucagon, gut glucagon, vasoactive intestinal polypeptide and secretin with liver and fat cell plasma membranes: binding of specific sites and stimulation of adenylate cyclase, Endocrinology, 96 (1974) 713-721. 3 Cramer, H., Hammers, R., Maier, P . and Schindler, H., Cyclic 3 /,5 '-adenos ine monophosphate in the choroid plexus: stimulation by cholera toxin , Biochem. Bioph ys, Res. Cornmun ., 84 (1978) 1031-1037. 4 Edvinsson, 1., HAkanson, R., Lindvall , M., Owman , Ch . and Svensson, K.-G., Ultrastructural and biochemical evidence for a sympathetic neural influence on the choroid plexus, Exp . Neurol., 48 (1975) 241-251. 5 Edvinsson, 1., Nielsen, K.C., Owrnan, Ch, and West, K.A., Adrenergic innervation of the mammalian choroid plexus, Arner. J . Anat., 139 (1974) 299-308. 6 Edvinsson, 1., Owman, Ch., Rosengren, E. and West, K.A ., Concentration of noradrenaline in pial vessels, choroid plexus, and iris during two weeks after sympathetic ganglionectomy or decentralization, Acta Physio!. Scand ., 85 (1972) 201-206. 7 Frandsen, E.K., Krishna, G.A. and Said, S.I., Vasoactive intestinal polypeptide promotes cyclic adenosine 3'-5'-monophosphate accumulation in guinea-pig trachea, Brit. 1. Pharmacol., 62 (1978) 367-369 . 8 Gilman, A.G. , A protein binding assay for adenosine 3' -5' -cyclic monopho sphate, Pro c, Nat. Acad , Sci. USA, 67 (1970) 305-312 . 9 Hammers , R. , Clarenbach, P ., Lindl, T . and Crame r, H ., Uptake and metabolism of cyclic AMP in rabbit choroid plexus in vitro , Neuropharmacology. 16 (1977) 135-141. 10 Laburthe, M. , Prieto , J .C., Amiranoff, B., Dupont, C ., Hu i Bon Hoa, D. and Rosselin, G. , Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 2. Characterization and structural requi rements of the stimulatory effect of vasoacti ve intestinal peptide on production of adenosine 3 '-5'-monophosphate, Europ. J. Biochern ., 96 (1979) 239-248.
157 II Lindvall, M., Fluorescence histochemical study on regional differences in the sympathetic nerve sup-
ply of the choroid plexus from various laboratory animals, Cell Tiss. Res., 198 (1979) 261-267. 12 Lindvall, M., Alumets, J., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Hanko, J., Owman, Ch., Schaffalitzky de Muckadell, O.B. and Sundler, F., Peptidergic (VIP) nerves in the mammalian choroid plexus, Neurosci, Lett., 9 (1978) 77-82. 13 Lindvall, M., Edvinsson, L. and Owman, Ch., Sympathetic nervous control of cerebrospinal fluid production from the choroid plexus, Science, 201 (1978) 176-178. 14 Lindvall, M., Edvinsson, L. and Owman, Ch., Effect of sympathomimetic drugs and corresponding receptor antagonists on the rate of cerebrospinal fluid production, Exp. Neuro!., 64 (1979) 132-145. 15 Lindvall, M., Owman, Ch. and Winbladh, B., Sympathetic influence on transport functions in the choroid plexus of rabbit and rat, Brain Res., 223 (1981) 160-164. 16 Lindvall, M., Owrnan , Ch. and Windbladh, B., Sympathetic influence on sodium-potassium activated adenosine triphosphatase activity of rabbit and rat choroid plexus, Brain Res. Bull., 9 (1982) 761-763. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, 1. Bio!. Chem., 193 (1951) 265-275. 18 Nathanson, J .A., I3-Adrenergic-sensitive adenylate cyclase in secretory cells of choroid plexus, Science, 204 (1979) 843-844. 19 Nathanson, J .A., I3-Adrenergic-sensitive adenylate cyclase in choroid plexus: properties and cellular localization, Molecu!. Pharmacol., 18 (1980) 199-209. 20 Pershing, L.K. and Johansson, C.E., Acidosis-induced enhanced activity of the Na-K exchange pump in the in vivo choroid plexus: an ontogenetic analysis of possible role in cerebrospinal fluid pH homeostasis, J. Neurochem., 38 (1982) 322-332. 21 Rudman, D., Hollins, B.M., Lewis, N.C. and Scott, J.W., Effects of hormones on 3',5'-cyclic adenosine monophosphate in choroid plexus, Amer. J. Physio!., 232 (1977) E353-E357. 22 Saito, Y. and Wright, E.M., Bicarbonate transport across the frog choroid plexus and its control by cyclic nucleotides, J. Physiol. (Lond.), 336 (1983) 635-648. 23 Tovey, K.C., Oldham, S.E. and Whelan, J.A.M., A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique, Clin. Chim. Acta, 56 (1974) 221-234.
153
NSL 03153
STIMULATION OF CYCLIC ADENOSINE 3' ,5'·MONOPHOSPHATE FORMATION IN RABBIT CHOROID PLEXUS BY .a-RECEPTOR AGONISTS AND VASOACTIVE INTESTINAL POLYPEPTIDE
MARIA LlNDVALL*, A.SA GUSTAFSON, PAVO HEDNER and CHRISTER OWMAN
Departments of Histology and Internal Medicine, University of Lund, Lund (Sweden) (Received June 27th, 1984; Revised version received November 23rd, 1984; Accepted November 26th, 1984)
Key words: choroid plexus - cyclic adenosine 3' ,5 '-monophosphate - {j-receptor agonists - vasoactive intestinal polypeptide
The formation of cyclic adenosine 3',5' -monophosphate (cAMP) was determined in blood-free choroid plexus homogenates from all ventricles of rabbit using a competitive protein binding technique. Previous stimulation of the intact plexus tissue in vitro with sympathomimetic drugs or vasoactive intestinal polypeptide (VIP) leads to increased local synthesis of cAMP. Tests with selective (3-receptor agonists and antagonists suggested that {jt-receptors predominate, which is consistent with studies of sympathomimetic effects on cerebrospinal fluid production in vivo.
The choroid plexus is innervated by adrenergic nerves [5, 11] originating in the superior cervical sympathetic ganglia [6, 11]. The nerve terminals are associated not only with the blood vessels but also with the secretory epithelium of the plexus [4]. Stimulation of the choroidal sympathetic nerves markedly inhibits the formation of cerebrospinal fluid (CSF), whereas the production rate is enhanced following denervation [13]. Pharmacological studies have suggested that the sympathetically mediated inhibition involves both .a-adrenergic receptors on the epithelial cells and ce-adrenoceptors in the vascular bed of the choroid plexus [14]. Studies of transport functions both in vitro [15, 16] and in vivo [20] have confirmed the direct functional influence of the sympathetic system on the secretory epithelium. It has recently been found by immunohistochemistry that the choroid plexus is also innervated by nerve fibers containing vasoactive intestinal polypeptide (VIP) [12]. Adrenergic receptors, particularly of the .a-type, are frequently associated with membrane-bound adenylate cyclase which, following activation, leads to the intracellular synthesis of cyclic adenosine 3' ,5' -monophosphate, referred to as cyclic AMP (cAMP) (cf. ref. 18). VIP also stimulates cAMP formation in several tissues *Author for correspondence at: Department of Histology, Biskopsgatan 5, S-223 62 Lund, Sweden.
154
[1, 2, 7, 10]. The present study on rabbits was undertaken to elucidate whether sympathomimetic stimulation or VIP leads to increased local synthesis of cAMP as a further means to define the functional role of certain adrenergic and peptidergic mechanisms in the choroid plexus. The experiments were performed in 23 randomly pigmented rabbits of either sex weighing 2.5-3.5 kg. They were fed with standard pellet food and tap water ad libitum. The animals were killed by perfusion with 0.9% saline through the left ventricle of the heart under sodium pentobarbital (Mebumal; ACO, Sweden) anesthesia (20 rug/kg i.v.), The amount of saline used in order to obtain completely blood-free plexus tissue was 100 ml. Choroid plexus tissue from each half of the brain including all ventricles was immediately dissected out. Each sample thus contained the plexus from the lateral ventricle and half of the plexus tissue from the third and fourth ventricles. The two batches of material from each animal were put in separate tubes containing 1 ml Krebs-Ringer bicarbonate buffer solution supplied with 10 mM glucose for preincubation during 30 min. Susequent incubation during 10 min was carried out in fresh medium provided with 1 mM theophylline (Sigma). Both incubation steps were performed at 37°C during aeration with a mixture of 9511,70 02-5010 CO 2, After finishing incubation, the tissue was transferred to ice-cold 10% trichloroacetic acid (TCA) for homogenization and the precipitated material was removed by centrifugation at 2500 g in 4°C for 30 min. As a technical control, the supernatant was supplied with 5000 cpm [3H]cAMP (2,8-[3H]adenosine 3',5 -cyclic phosphate, ammonium salt, 30-50 Cilmmol; New England Nuclear) for later estimation of recovery of endogenous cAMP, and put on a Dowex SOW column with 1.5 ml bed volume. cAMP was eluted with distilled water, lyophilized to dryness and resuspended in 500 Itl distilled water for its determination by a modified Gilman [8] method developed by Tovey et al. [23] using charcoal for separation of bound from free cAMP. Recovery was determined by counting a 50 Itl aliquot of the resuspended material. Tissue protein was determined in each tissue sample by the colorimetric method of Lowry et al. [17]. Differences between the mean values of cAMP concentrations in the plexus tissue (expressed as pmol cAMP Img tissue protein) in the different experimental groups were evaluated statistically with Student's t-test. The mean basal level of cAMP in the rabbit choroid plexus after a 10 min incubation period was 48 ± 4 (S.E.M.) pmol/mg protein. The results of the drug effects are summarized in Fig. 1. The (12-adrenoceptor selective agonist, terbutaline (Bricanyl; Draco, Sweden), caused a dose-dependent increase in the cAMP formation. A more pronounced effect was obtained in the presence of the t31-selective agonist H80/62 (Hassle, Sweden). Blockade of the (11-type receptors with practolol (Eraldine; ICI) markedly reduced the effect of H80/62, whereas practolol in combination with terbutaline instead showed a tendency to increased cAMP formation. In the presence of 5 j.tg/ml highly purified porcine VIP (generous gift from Professor Viktor Mutt, Dept. Biochem., Karolinska Institute, Stockholm, Sweden) the
155
cAMP formation was enhanced to the same degree as achieved with 10- 6 M terbutaline in the incubation medium. The choroid plexus is equipped with a cAMP-generating system, suggesting a physiological role of cAMP in the function of this structure [9, 22]. Initial studies failed to activate this system in the rabbit plexus by sympathomimetic stimulation [9, 21], although isoproterenol and epinephrine, as well as norepinephrine, were all able to influence the cAMP content in the rat's choroid plexus. Later, isoproterenol was found to produce an increase in cAMP also in the rabbit plexus tissue [3, 18]. On the basis of the potency rate for the efficiency of various sympathomimetic compounds to stimulate the formation of cAMP in combination with several more or less selective antagonists, Nathanson [19] concluded that the {J-receptor mediating the effect in the feline choroid plexus was of the f32-type. The present findings on rabbit, on the other hand, rather suggest that {JI-receptors predominate. This is consistent with observations on the CSF production rate in the rabbit in vivo,
Treatment
o
cAMP (pmoles/mg protein)
I
Untreated control Terbutaline (l0·6Ml Terbutaline 110"M) 6M)
Terbutaline 110· + practolol (lO"M)
___
50 I
100
150
I
I
---J~ (13) (4)** (5)***
~(4)***
H 80/62 (10- 6M)
(5)***
H 80/62 OO·'M)
H 80/62 (1 0-6M ) +
practolol (lO"M)
(6)***
~(4)***
VIP (SI-lg/ml) Fig. 1. Levels of cAMP determined by protein binding in homogenized rabbit choroid plexus after previous incubations of intact plexus tissue for 10 min in the presence of different sympathomimetic agonists (terbutaline, H80/62), antagonist (practolol) and vasoactive intestinal polypeptide (VIP). Differences from mean control values (± S.E.M.) according to Student's I-test: **0.001< P< 0.01, **"P< 0.001. Number of determinations within parentheses. Controls, unfilled bar; agonists, filled bars; antagonist, hatched bars.
156
where the strong sympathetic inhibition [13J seems to involve {1-receptors in the plexus epithelium of mainly the (31-type [14J. VIP has previously been shown to stimulate cAMP formation in several tissues [1,2, 7, 10J. The formation of cAMP in the rabbit choroid plexus is also stimulated by VIP. Immunohistochemical studies have shown VIP-containing nerve fibers to be present in the structure, particularly around plexus blood vessels, which dilate in response to VIP in vitro [12J. However, considerable species differences were found . In the choroid plexus from pig, a moderate number of VIP-containing nerves was found not only with a perivascular distribution, but also in close association with the plexus epithelium [12J. In contrast, VIP nerves were few in rabbit choroid plexuses [12]. It is thus not unlikely that the VIP-induced stimulation of cAMP formation reflects a functional role of VIP in the choroid plexus, where the putative receptors mediating the peptidergic effect are not necessarily related to the presence of VIP-ergic nerve fibers in the rabbit. Supported by the Swedish Medical Research Council (Grant 04X-732) and by AB Draco, Lund, Sweden.
1 Ahren, B., Alumets, J ., Ericsson, M., Fahrenkrug, L. , HAkanson, R., Hedner, P., Loren, 1., Melander A. , Rerup, C. and Sundler , F., VIP occurs in intrathyroidal nerves and stimulates thyroid hormone secretion , Nature (Lond.), 287 (1980) 343-345. 2 Bataille, D., Freychet, P. and Rosselin, G., Interactions of glucagon, gut glucagon, vasoactive intestinal polypeptide and secretin with liver and fat cell plasma membranes: binding of specific sites and stimulation of adenylate cyclase, Endocrinology, 96 (1974) 713-721. 3 Cramer, H., Hammers, R., Maier, P . and Schindler, H., Cyclic 3 /,5 '-adenos ine monophosphate in the choroid plexus: stimulation by cholera toxin , Biochem. Bioph ys, Res. Cornmun ., 84 (1978) 1031-1037. 4 Edvinsson, 1., HAkanson, R., Lindvall , M., Owman , Ch . and Svensson, K.-G., Ultrastructural and biochemical evidence for a sympathetic neural influence on the choroid plexus, Exp . Neurol., 48 (1975) 241-251. 5 Edvinsson, 1., Nielsen, K.C., Owrnan, Ch, and West, K.A., Adrenergic innervation of the mammalian choroid plexus, Arner. J . Anat., 139 (1974) 299-308. 6 Edvinsson, 1., Owman, Ch., Rosengren, E. and West, K.A ., Concentration of noradrenaline in pial vessels, choroid plexus, and iris during two weeks after sympathetic ganglionectomy or decentralization, Acta Physio!. Scand ., 85 (1972) 201-206. 7 Frandsen, E.K., Krishna, G.A. and Said, S.I., Vasoactive intestinal polypeptide promotes cyclic adenosine 3'-5'-monophosphate accumulation in guinea-pig trachea, Brit. 1. Pharmacol., 62 (1978) 367-369 . 8 Gilman, A.G. , A protein binding assay for adenosine 3' -5' -cyclic monopho sphate, Pro c, Nat. Acad , Sci. USA, 67 (1970) 305-312 . 9 Hammers , R. , Clarenbach, P ., Lindl, T . and Crame r, H ., Uptake and metabolism of cyclic AMP in rabbit choroid plexus in vitro , Neuropharmacology. 16 (1977) 135-141. 10 Laburthe, M. , Prieto , J .C., Amiranoff, B., Dupont, C ., Hu i Bon Hoa, D. and Rosselin, G. , Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 2. Characterization and structural requi rements of the stimulatory effect of vasoacti ve intestinal peptide on production of adenosine 3 '-5'-monophosphate, Europ. J. Biochern ., 96 (1979) 239-248.
157 II Lindvall, M., Fluorescence histochemical study on regional differences in the sympathetic nerve sup-
ply of the choroid plexus from various laboratory animals, Cell Tiss. Res., 198 (1979) 261-267. 12 Lindvall, M., Alumets, J., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Hanko, J., Owman, Ch., Schaffalitzky de Muckadell, O.B. and Sundler, F., Peptidergic (VIP) nerves in the mammalian choroid plexus, Neurosci, Lett., 9 (1978) 77-82. 13 Lindvall, M., Edvinsson, L. and Owman, Ch., Sympathetic nervous control of cerebrospinal fluid production from the choroid plexus, Science, 201 (1978) 176-178. 14 Lindvall, M., Edvinsson, L. and Owman, Ch., Effect of sympathomimetic drugs and corresponding receptor antagonists on the rate of cerebrospinal fluid production, Exp. Neuro!., 64 (1979) 132-145. 15 Lindvall, M., Owman, Ch. and Winbladh, B., Sympathetic influence on transport functions in the choroid plexus of rabbit and rat, Brain Res., 223 (1981) 160-164. 16 Lindvall, M., Owrnan , Ch. and Windbladh, B., Sympathetic influence on sodium-potassium activated adenosine triphosphatase activity of rabbit and rat choroid plexus, Brain Res. Bull., 9 (1982) 761-763. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, 1. Bio!. Chem., 193 (1951) 265-275. 18 Nathanson, J .A., I3-Adrenergic-sensitive adenylate cyclase in secretory cells of choroid plexus, Science, 204 (1979) 843-844. 19 Nathanson, J .A., I3-Adrenergic-sensitive adenylate cyclase in choroid plexus: properties and cellular localization, Molecu!. Pharmacol., 18 (1980) 199-209. 20 Pershing, L.K. and Johansson, C.E., Acidosis-induced enhanced activity of the Na-K exchange pump in the in vivo choroid plexus: an ontogenetic analysis of possible role in cerebrospinal fluid pH homeostasis, J. Neurochem., 38 (1982) 322-332. 21 Rudman, D., Hollins, B.M., Lewis, N.C. and Scott, J.W., Effects of hormones on 3',5'-cyclic adenosine monophosphate in choroid plexus, Amer. J. Physio!., 232 (1977) E353-E357. 22 Saito, Y. and Wright, E.M., Bicarbonate transport across the frog choroid plexus and its control by cyclic nucleotides, J. Physiol. (Lond.), 336 (1983) 635-648. 23 Tovey, K.C., Oldham, S.E. and Whelan, J.A.M., A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique, Clin. Chim. Acta, 56 (1974) 221-234.