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Modulation of adenylyl cyclase activity by baclofen in the developing rat brain: difference between cortex, thalamus and hippocampus
Neuroscience Letters 330 (2002) 9–12 www.elsevier.com/locate/neulet
Modulation of adenylyl cyclase activity by baclofen in the developing rat brain: difference between cortex, thalamus and hippocampus Lucie Hejnova´ a,b, Ivanna Ihnatovych b,c, Jiri Novotny a,b, Hana Kubova´ c, Pavel Maresˇ c, Petr Svoboda a,b,* a
Department of Biochemistry of Membrane Receptors, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic b Department of Physiology, Faculty of Natural Sciences, Charles University, 120 00 Prague 2, Czech Republic c Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic Received 25 March 2002; received in revised form 19 June 2002; accepted 19 June 2002
Abstract Ontogenetic changes in the levels of GABAB receptors and their ability to modulate adenylyl cyclase (AC) activity were analyzed in rat cortex, thalamus and hippocampus. The relative numbers of GABAB receptors (measured as saturable, high-affinity [ 3H](2)baclofen binding sites) in cortex and thalamus were high already at postnatal day 1 (PD 1) and they reached a maximum at PD 25 and PD 12, respectively. There were no detectable high-affinity [ 3H](2)baclofen binding sites in hippocampus between birth and PD 12 and low-affinity [ 3H](2)baclofen binding attained at PD 12 did not change in adulthood (PD 90). Whereas GTP-stimulated AC activity in cortex and thalamus was depressed by baclofen, it was enhanced in hippocampus. These data indicate that the inhibitory effect of baclofen on AC in cortex and thalamus is primarily mediated through the a subunits of Gi/Go proteins. The stimulatory effect of baclofen in hippocampus may be explained by engagement of Gbg subunits. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GABAB receptors; Baclofen; Adenylyl cyclase; Developing rat brain
The GABAergic signalling system is a major inhibitory system in the brain. GABA exerts its action through two different types of membrane receptors – ionotropic (GABAA and GABAC) and metabotropic (GABAB). GABAB receptors were described pharmacologically as bicuculline-insensitive metabotropic GABA receptors that can be activated by baclofen [7]. The wide distribution of these receptors in CNS, pharmacological heterogeneity as well as multiple functional roles of GABAB receptors attest to their crucial importance in brain signalling [3,20]. Signals from activated GABAB receptors are transmitted through pertussis toxin-sensitive G proteins of the Gi/Go family [12,14]. These proteins are engaged in activation of inwardly rectifying potassium channels, inhibition of
* Corresponding author. Department of Biochemistry of Membrane Receptors, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic. Tel.: 1420-2-41062533; fax: 1420-2-41062488. E-mail address: [email protected] (P. Svoboda).
voltage-gated calcium channels and negative regulation of adenylyl cyclase (AC) activity [6,11]. Although activation of Gi proteins is usually associated with inhibition of AC activity, it has also been shown that in the presence of activated Gs, Gi proteins can mediate stimulation of type II and IV of AC. This stimulation is mediated by Gbg subunits [18]. Thus, besides inhibition of basal as well as forskolinstimulated AC activity [15,21], activation of GABAB receptors by baclofen may result in stimulation of this enzyme activity [16]. The present work analyzed GABAB (baclofen)-mediated effects on AC activity in the developing rat cortex, thalamus and hippocampus. The number of GABAB receptors was determined in parallel. Male Wistar rats were killed by decapitation at postnatal day (PD) 1, 7, 12, 18, 25 or 90. Cerebral cortex, thalamus and hippocampus were rapidly isolated, frozen in liquid nitrogen and stored at 270 8C. After thawing, the brain tissues were minced with a razor blade and homogenized in 10 ml of TMSDE buffer (50 mM Tris–HCl, 6 mM MgCl2,
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 72 1- 8
L. Hejnova´ et al. / Neuroscience Letters 330 (2002) 9–12
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Table 1 GABAB receptor binding in immature (PD 12) and adult (PD 90) rat brain a Cortex
PD 12 PD 90
Thalamus
Hippocampus
KD
Bmax
KD
Bmax
KD
Bmax
116 ^ 21 144 ^ 35
1200 ^ 225 1001 ^ 189
167 ^ 42 304 ^ 38
992 ^ 167 600 ^ 88
* *
* *
a KD and Bmax values are expressed as nmol l 21 (nM) and fmol mg 21 protein, respectively. Each value represents the mean ^ SEM obtained from three separate experiments. *, non-saturable up to a 240 nM concentration of [ 3H](2)baclofen.
75 mM sucrose, 1 mM DTT, 1 mM EDTA; pH 7.6) per gram wet weight by using a Potter–Elvehjem homogenizer (Teflon-glass). Unbroken cells and nuclei were removed by centrifugation at 600 £ g for 5 min (4 8C). The postnuclear supernatants were subjected to centrifugation at 48,000 £ g for 30 min at 4 8C. The membrane sediments were resuspended in buffer A (50 mM Tris–HCl; pH 7.4) and centrifuged as above. This washing procedure was repeated six times in total. The final membrane sediments were resuspended in buffer A (for AC assay) or in radioligand binding assay buffer B (50 mM Tris–HCl, 2.5 mM CaCl2; pH 7.4). The [ 3H](2)baclofen binding assay was performed according to Robinson et al. [17] in a total volume of 0.2 ml of buffer B supplemented either by 20 nM [ 3H](2)baclofen (one-point assay) or 20–240 nM [ 3H](2)baclofen (saturation binding assay). The binding reaction was initiated by the addition of 0.15–0.2 mg membrane protein and carried out for 20 min at 25 8C. The reaction was terminated by addition of 3 ml of ice-cold buffer A and rapid filtration through Whatman GF/C glass filters on a Brandel Cell Harvestor. The filters were then washed twice with 3 ml of buffer A. Non-specific binding was defined as that remaining in the presence of 1 mM (^)baclofen. Radioactivity retained on the filters was measured by liquid scintillation. The activity of AC was determined as described previously [8]. In brief, membranes (50–100 mg protein) were incubated for 20 min at 32 8C in a total volume of 0.1 ml of reaction mix containing 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 50 units/ml pyruvate kinase, 10 mM potassium phosphoenolpyruvate, 0.2 mg/ ml bovine serum albumin, 0.2 mM 3-isobutyl-1-methylxanthine, 0.1 mM cAMP, 10,000 cpm per sample of [ 3H]cAMP and 0.4 mM ATP 1 [a- 32P]ATP (about 1 £ 10 6 cpm per sample). The assay was run in the presence of GTP (10 mM) and with or without baclofen (10 mM). The reaction was stopped by adding 0.2 ml of 0.5 M HCl and heating for 5 min at 100 8C. The cyclic AMP was separated by alumina column chromatography and the detected amount of [ 32P]cAMP was corrected according to the recovery of added [ 3H]cAMP. The number and affinity of GABAB receptors in immature (PD 12) and adult (PD 90) rat brain were studied first by saturation binding assay using 20–240 nM [ 3H](2)baclofen.
Saturable, high-affinity [ 3H](2)baclofen binding sites were detected in membrane samples of immature as well as adult rat cortex and thalamus. Whereas there was no significant difference in the affinity and number of GABAB receptors in cortex of 12- and 90-day-old rats, KD values determined in thalamus at PD 12 were lower than those detected at PD 90 and the reverse was true for Bmax values (Table 1). Surprisingly, no saturable [ 3H](2)baclofen binding was found in hippocampus up to a 240 nM concentration of the radioligand. The density of an overall population of GABAB receptors was determined by ‘one-point’ radioligand binding assay using a 20 nM concentration of [ 3H](2)baclofen. We checked by calculation (B ¼ Bmax L=K D 1 L) that the observed developmental changes in GABAB receptors were not significantly distorted by using this low radioligand concentration. The high-affinity [ 3H](2)baclofen binding in cortex and thalamus was clearly detectable already shortly after birth (PD 1) and this binding increased largely during the first 2–3 weeks of postnatal life (Fig. 1). The maximum levels were reached at PD 25 and PD 12 in cortex and thalamus, respectively. Next development was associated with a pronounced decrease in the density of [ 3H](2)baclofen binding sites. There was apparently no high-affinity binding before PD 12 in hippocampus. Afterwards, a slight [ 3H](2)baclofen binding was achieved and it
Fig. 1. Developmental changes in the density of GABAB receptors in various brain regions. GABAB receptors were determined by ‘one-point assay’ radioligand binding study using 20 mM [ 3H](2)baclofen in membrane preparations of rat cortex (A), thalamus (O) and hippocampus (W). Values represent means ^ SEM of three separate experiments performed in triplicate.
L. Hejnova´ et al. / Neuroscience Letters 330 (2002) 9–12
11
Fig. 2. Baclofen modulation of GTP-stimulated AC activity in the developing rat brain. The activity of AC in membrane samples of cortex (A), thalamus (B) and hippocampus (C) was determined in the presence of 10 mM GTP ^ 10 mM baclofen (empty and solid columns, respectively). Values represent the mean ^ SEM of three separate experiments performed in duplicate. The statistical significance of the baclofen effect was determined by two-tailed Student’s t-test, *P , 0:05.
did not change in the course of further development (Fig. 1). These binding sites exhibit a very low affinity, since a 1 mM concentration of unlabelled baclofen was used to determine non-specific binding. Signal transduction via metabotropic GABAB receptors was the subject of considerable experimental interest during the past decade [9]. Ontogenetic distribution of GABAB receptors in brain tissues was studied by [ 3H]GABA radioligand binding studies [5], receptor autoradiography [19], immunoblotting and immunohistochemistry [10]. Our present measurements disclosed a different distribution of GABAB receptors in three analyzed brain regions. The number of high-affinity [ 3H](2)baclofen binding sites increased progressively from birth till PD 25 (PD 12) in cortex (thalamus) and subsequently declined to adult levels. A similar developmental pattern of GABAB receptors (maximum level at PD 14 in cortex) was previously described [19]. In contrast to cortex and thalamus, we were unable to find any saturable, high-affinity [ 3H](2)baclofen binding sites in hippocampus during the first postnatal week and further development was associated only with a minor invariable radioligand binding. Contrarily, it has been reported that GABAB receptor binding peaks already at PD 3 or PD 7 in CA3 and CA1 areas of hippocampus [19]. These observations might indicate unequal distribution of these receptors in different hippocampal regions. Another [ 3H]GABA receptor binding study demonstrated lower density of GABAB sites in hippocampal tissue from 14- to 17-day-old rat pups than in adult animals [5]. These discrepancies may be due to pharmacologically distinct populations of GABAB receptors exhibiting different sensitivity and binding capacity towards different GABAB ligands [4]. The different pharmacological properties of these receptor sites, in turn, may be caused by the structural heterogeneity of GABAB receptors [13]. Next we examined the influence of baclofen on AC activity in all tested brain regions. Relatively high GTP-dependent AC activity was detected already at PD 1 in cortex, thalamus as well as in hippocampus (120, 250 and 200 pmol cAMP min 21 mg 21, respectively) and it increased dramati-
cally during the early postnatal development to reach the maximum levels at PD 12 (Fig. 2). Subsequently, AC activity gradually decreased to the adult level (PD 90), which was lower than that measured at PD 1 (Fig. 2). Baclofen significantly affected AC activity, but its influence was much smaller than the general ontogeneticallyinduced alterations in AC activity (Fig. 2). The effect of baclofen exhibited a clear resemblance to characteristics of [ 3H](2)baclofen binding. Baclofen at a 10 mM concentration exerted significant inhibition of GTP-stimulated AC activity in cortex at PD 12 and this inhibition was further increased with maturation – no effect at PD 1 and 7, inhibition by 24, 29, 37 and 46% at PD 12, 18, 25 and 90, respectively. Similar reduction of AC activity was observed in thalamus. Contrarily, GTP-dependent AC activity in hippocampal membranes prepared from 12- and 18-day-old rats was markedly enhanced by baclofen (Fig. 2). These differential modulatory effects of baclofen in the three tested brain regions were clearly dependent on the stage of ontogenetic development. Our previous study dealing with ontogenetic development of the AC signalling in rat brain provided similar results: baclofen reduced forskolin-stimulated AC in cortex and the extent of this inhibition increased with maturation. Inhibition of forskolin-stimulated AC was also observed in thalamus [8]. Contrarily, in hippocampus, baclofen did not affect forskolin-stimulated AC activity at any of the age intervals between PD 1 and PD 90. It was suggested that stimulation of 5-HT and GABAB receptors might potentiate b-adrenergic responses signalled through AC in hippocampus [1] and a region-specific enhancement of basal and neurotransmitter-stimulated AC through activation of GABAB receptors has recently been described in the rat olfactory bulb [16]. These observations are consistent with the evidence indicating that Gi-linked receptors can enhance the ability of Gs to stimulate type II and IV of AC [18]. Discrete distribution of both these AC isozymes in hippocampal formation was revealed by immunohistochemical labelling [2]. In our previous study we did not observe any substantial differences in the ontogenetic expression of AC in cortex, thalamus and hippocampus
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L. Hejnova´ et al. / Neuroscience Letters 330 (2002) 9–12
[8]. Adult levels of AC achieved around PD 12 and 18 were maintained in adulthood. Similarly, there were no significant discontinuities in the expression of Gs and Gi/Go proteins in these brain regions. Despite that, AC activity exhibited a pronounced maximum at PD 12, which is in agreement with our current results. Taking into consideration all these facts, we think that a specific modulation of AC activity in hippocampus can not be explained only on the basis of quantitation of AC molecules and G proteins. We would rather suppose that specific organization of neurotransmission systems in hippocampus might cause differential regulation of AC in this region, which represents a crucial brain structure for memory formation. The role of cAMP in long-term potentiation has been well documented [22]. The positive coupling of GABAB receptors to AC may be explained by the co-localization of GABAB receptors, Gi/Go proteins, Gs-linked receptors, Gs and AC molecules at specific synaptic or membrane-domain sites [16], where all these molecules co-exist and interact by functionally significant protein–protein interactions. It can not be ruled out, however, that the population of GABAB receptors in hippocampus is pharmacologically distinct from that detected in the other brain regions. This notion is supported by our present finding of a low level of low-affinity [ 3H](2)baclofen binding sites in hippocampus, which is in contrast to cortex and thalamus. It can be speculated that different levels of GABAB receptors with distinct pharmacological properties might in certain conditions exercise their regulatory function towards AC differentially. This work was supported by grants from the Grant Agency of the Czech Republic (309/99/207 and 309/01/ 0255) and by the Ministry of Education of the Czech Republic (projects LN00A026 and 11300003). [1] Andrade, R., Enhancement of b-adrenergic responses by Gi-linked receptors in rat hippocampus, Neuron, 10 (1993) 83–88. [2] Baker, L.P., Nielsen, M.D., Impey, S., Hacker, B.M., Poser, S.W., Chan, M.Y. and Storm, D.R., Regulation and immunohistochemical localization of bg-stimulated adenylyl cyclases in mouse hippocampus, J. Neurosci., 19 (1999) 180–192. [3] Bowery, N.G., GABAB receptor pharmacology, Annu. Rev. Pharmacol. Toxicol., 33 (1993) 109–147. [4] Cunningham, M.D. and Enna, S.J., Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain, Brain Res., 720 (1996) 220–224. [5] Garant, D., Sperber, E. and Moshe, S., The density of GABAB binding sites in the substantia nigra is greater in rat pups than in adults, Eur. J. Pharmacol., 214 (1992) 75–78. [6] Hill, D.R., GABAB receptor modulation of adenylate cyclase activity in rat brain slices, Br. J. Pharmacol., 84 (1985) 249– 257. [7] Hill, D.R. and Bowery, N.G., [ 3H]baclofen and [ 3H]GABA
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bind to bicuculline-insensitive GABAB sites in rat brain, Nature, 290 (1981) 149–152. Ihnatovych, I., Novotny, J., Haugvicova, R., Bourova, L., Mares, P. and Svoboda, P., Ontogenetic development of the G protein-mediated adenylyl cyclase signalling in rat brain, Brain Res. Dev. Brain Res., 133 (2002) 69–75. Kerr, D.I. and Ong, J., GABAB receptors, Pharmacol. Ther., 67 (1995) 187–246. Malitschek, B., Ruegg, D., Heid, J., Kaupmann, K., Bittiger, H., Frostl, W., Bettler, B. and Kuhn, R., Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain, Mol. Cell. Neurosci., 12 (1998) 56–64. Misgeld, U., Bijak, M. and Jarolimek, W., A physiological role for GABAB receptors and the effects of baclofen in the mammalian central nervous system, Prog. Neurobiol., 46 (1995) 423–462. Morishita, R., Kato, K. and Asano, T., GABAB receptors couple to G proteins Go, Go* and Gi1 but not to Gi2, FEBS Lett., 271 (1990) 231–235. Ng, G.Y., Bertrand, S., Sullivan, R., Ethier, N., Wang, J., Yergey, J., Belley, M., Trimble, L., Bateman, K., Alder, L., Smith, A., McKernan, R., Metters, K., O’Neill, G.P., Lacaille, J.C. and Hebert, T.E., Gamma-aminobutyric acid type B receptors with specific heterodimer composition and postsynaptic actions in hippocampal neurons are targets of anticonvulsant gabapentin action, Mol. Pharmacol., 59 (2001) 144–152. Nishikawa, M., Hirouchi, M. and Kuriyama, K., Functional coupling of Gi subtype with GABAB receptor/adenylyl cyclase system: analysis using a reconstituted system with purified GTP-binding protein from bovine cerebral cortex, Neurochem. Int., 31 (1997) 21–25. Nishikawa, M. and Kuriyama, K., Functional coupling of gamma-aminobutyric acid GABAB receptor with adenylate cyclase system: effect of phaclofen, Neurochem. Int., 14 (1989) 85–90. Olianas, M.C. and Onali, P., GABAB receptor-mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb, Br. J. Pharmacol., 126 (1999) 657–664. Robinson, T.N., Cross, A.J., Green, A.R., Toczek, J.M. and Boar, B.R., Effects of the putative antagonists phaclofen and d-aminovaleric acid on GABAB receptor biochemistry, Br. J. Pharmacol., 98 (1989) 833–840. Tang, W.J., Iniguez-Lluhi, J.A., Mumby, S. and Gilman, A.G., Regulation of mammalian adenylyl cyclases by Gprotein a and bg subunits, Cold Spring Harb. Symp. Quant. Biol., 57 (1992) 135–144. Turgeon, S.M. and Albin, R.L., Postnatal ontogeny of GABAB binding in rat brain, Neuroscience, 62 (1994) 601– 613. Wojcik, W.J. and Holopainen, I., Role of central GABAB receptors in physiology and pathology, Neuropsychopharmacology, 6 (1992) 201–214. Xu, J. and Wojcik, W.J., Gamma aminobutyric acid B receptor-mediated inhibition of adenylate cyclase in cultured cerebellar granule cells: blockade by islet-activating protein, J. Pharmacol. Exp. Ther., 239 (1986) 568–573. Yu, T.P., McKinney, S., Lester, H.A. and Davidson, N., Gamma-aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3-CA1 synapses, Proc. Natl. Acad. Sci. USA, 98 (2001) 5264–5269.
Modulation of adenylyl cyclase activity by baclofen in the developing rat brain: difference between cortex, thalamus and hippocampus Lucie Hejnova´ a,b, Ivanna Ihnatovych b,c, Jiri Novotny a,b, Hana Kubova´ c, Pavel Maresˇ c, Petr Svoboda a,b,* a
Department of Biochemistry of Membrane Receptors, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic b Department of Physiology, Faculty of Natural Sciences, Charles University, 120 00 Prague 2, Czech Republic c Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic Received 25 March 2002; received in revised form 19 June 2002; accepted 19 June 2002
Abstract Ontogenetic changes in the levels of GABAB receptors and their ability to modulate adenylyl cyclase (AC) activity were analyzed in rat cortex, thalamus and hippocampus. The relative numbers of GABAB receptors (measured as saturable, high-affinity [ 3H](2)baclofen binding sites) in cortex and thalamus were high already at postnatal day 1 (PD 1) and they reached a maximum at PD 25 and PD 12, respectively. There were no detectable high-affinity [ 3H](2)baclofen binding sites in hippocampus between birth and PD 12 and low-affinity [ 3H](2)baclofen binding attained at PD 12 did not change in adulthood (PD 90). Whereas GTP-stimulated AC activity in cortex and thalamus was depressed by baclofen, it was enhanced in hippocampus. These data indicate that the inhibitory effect of baclofen on AC in cortex and thalamus is primarily mediated through the a subunits of Gi/Go proteins. The stimulatory effect of baclofen in hippocampus may be explained by engagement of Gbg subunits. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GABAB receptors; Baclofen; Adenylyl cyclase; Developing rat brain
The GABAergic signalling system is a major inhibitory system in the brain. GABA exerts its action through two different types of membrane receptors – ionotropic (GABAA and GABAC) and metabotropic (GABAB). GABAB receptors were described pharmacologically as bicuculline-insensitive metabotropic GABA receptors that can be activated by baclofen [7]. The wide distribution of these receptors in CNS, pharmacological heterogeneity as well as multiple functional roles of GABAB receptors attest to their crucial importance in brain signalling [3,20]. Signals from activated GABAB receptors are transmitted through pertussis toxin-sensitive G proteins of the Gi/Go family [12,14]. These proteins are engaged in activation of inwardly rectifying potassium channels, inhibition of
* Corresponding author. Department of Biochemistry of Membrane Receptors, Institute of Physiology, Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic. Tel.: 1420-2-41062533; fax: 1420-2-41062488. E-mail address: [email protected] (P. Svoboda).
voltage-gated calcium channels and negative regulation of adenylyl cyclase (AC) activity [6,11]. Although activation of Gi proteins is usually associated with inhibition of AC activity, it has also been shown that in the presence of activated Gs, Gi proteins can mediate stimulation of type II and IV of AC. This stimulation is mediated by Gbg subunits [18]. Thus, besides inhibition of basal as well as forskolinstimulated AC activity [15,21], activation of GABAB receptors by baclofen may result in stimulation of this enzyme activity [16]. The present work analyzed GABAB (baclofen)-mediated effects on AC activity in the developing rat cortex, thalamus and hippocampus. The number of GABAB receptors was determined in parallel. Male Wistar rats were killed by decapitation at postnatal day (PD) 1, 7, 12, 18, 25 or 90. Cerebral cortex, thalamus and hippocampus were rapidly isolated, frozen in liquid nitrogen and stored at 270 8C. After thawing, the brain tissues were minced with a razor blade and homogenized in 10 ml of TMSDE buffer (50 mM Tris–HCl, 6 mM MgCl2,
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 72 1- 8
L. Hejnova´ et al. / Neuroscience Letters 330 (2002) 9–12
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Table 1 GABAB receptor binding in immature (PD 12) and adult (PD 90) rat brain a Cortex
PD 12 PD 90
Thalamus
Hippocampus
KD
Bmax
KD
Bmax
KD
Bmax
116 ^ 21 144 ^ 35
1200 ^ 225 1001 ^ 189
167 ^ 42 304 ^ 38
992 ^ 167 600 ^ 88
* *
* *
a KD and Bmax values are expressed as nmol l 21 (nM) and fmol mg 21 protein, respectively. Each value represents the mean ^ SEM obtained from three separate experiments. *, non-saturable up to a 240 nM concentration of [ 3H](2)baclofen.
75 mM sucrose, 1 mM DTT, 1 mM EDTA; pH 7.6) per gram wet weight by using a Potter–Elvehjem homogenizer (Teflon-glass). Unbroken cells and nuclei were removed by centrifugation at 600 £ g for 5 min (4 8C). The postnuclear supernatants were subjected to centrifugation at 48,000 £ g for 30 min at 4 8C. The membrane sediments were resuspended in buffer A (50 mM Tris–HCl; pH 7.4) and centrifuged as above. This washing procedure was repeated six times in total. The final membrane sediments were resuspended in buffer A (for AC assay) or in radioligand binding assay buffer B (50 mM Tris–HCl, 2.5 mM CaCl2; pH 7.4). The [ 3H](2)baclofen binding assay was performed according to Robinson et al. [17] in a total volume of 0.2 ml of buffer B supplemented either by 20 nM [ 3H](2)baclofen (one-point assay) or 20–240 nM [ 3H](2)baclofen (saturation binding assay). The binding reaction was initiated by the addition of 0.15–0.2 mg membrane protein and carried out for 20 min at 25 8C. The reaction was terminated by addition of 3 ml of ice-cold buffer A and rapid filtration through Whatman GF/C glass filters on a Brandel Cell Harvestor. The filters were then washed twice with 3 ml of buffer A. Non-specific binding was defined as that remaining in the presence of 1 mM (^)baclofen. Radioactivity retained on the filters was measured by liquid scintillation. The activity of AC was determined as described previously [8]. In brief, membranes (50–100 mg protein) were incubated for 20 min at 32 8C in a total volume of 0.1 ml of reaction mix containing 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 50 units/ml pyruvate kinase, 10 mM potassium phosphoenolpyruvate, 0.2 mg/ ml bovine serum albumin, 0.2 mM 3-isobutyl-1-methylxanthine, 0.1 mM cAMP, 10,000 cpm per sample of [ 3H]cAMP and 0.4 mM ATP 1 [a- 32P]ATP (about 1 £ 10 6 cpm per sample). The assay was run in the presence of GTP (10 mM) and with or without baclofen (10 mM). The reaction was stopped by adding 0.2 ml of 0.5 M HCl and heating for 5 min at 100 8C. The cyclic AMP was separated by alumina column chromatography and the detected amount of [ 32P]cAMP was corrected according to the recovery of added [ 3H]cAMP. The number and affinity of GABAB receptors in immature (PD 12) and adult (PD 90) rat brain were studied first by saturation binding assay using 20–240 nM [ 3H](2)baclofen.
Saturable, high-affinity [ 3H](2)baclofen binding sites were detected in membrane samples of immature as well as adult rat cortex and thalamus. Whereas there was no significant difference in the affinity and number of GABAB receptors in cortex of 12- and 90-day-old rats, KD values determined in thalamus at PD 12 were lower than those detected at PD 90 and the reverse was true for Bmax values (Table 1). Surprisingly, no saturable [ 3H](2)baclofen binding was found in hippocampus up to a 240 nM concentration of the radioligand. The density of an overall population of GABAB receptors was determined by ‘one-point’ radioligand binding assay using a 20 nM concentration of [ 3H](2)baclofen. We checked by calculation (B ¼ Bmax L=K D 1 L) that the observed developmental changes in GABAB receptors were not significantly distorted by using this low radioligand concentration. The high-affinity [ 3H](2)baclofen binding in cortex and thalamus was clearly detectable already shortly after birth (PD 1) and this binding increased largely during the first 2–3 weeks of postnatal life (Fig. 1). The maximum levels were reached at PD 25 and PD 12 in cortex and thalamus, respectively. Next development was associated with a pronounced decrease in the density of [ 3H](2)baclofen binding sites. There was apparently no high-affinity binding before PD 12 in hippocampus. Afterwards, a slight [ 3H](2)baclofen binding was achieved and it
Fig. 1. Developmental changes in the density of GABAB receptors in various brain regions. GABAB receptors were determined by ‘one-point assay’ radioligand binding study using 20 mM [ 3H](2)baclofen in membrane preparations of rat cortex (A), thalamus (O) and hippocampus (W). Values represent means ^ SEM of three separate experiments performed in triplicate.
L. Hejnova´ et al. / Neuroscience Letters 330 (2002) 9–12
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Fig. 2. Baclofen modulation of GTP-stimulated AC activity in the developing rat brain. The activity of AC in membrane samples of cortex (A), thalamus (B) and hippocampus (C) was determined in the presence of 10 mM GTP ^ 10 mM baclofen (empty and solid columns, respectively). Values represent the mean ^ SEM of three separate experiments performed in duplicate. The statistical significance of the baclofen effect was determined by two-tailed Student’s t-test, *P , 0:05.
did not change in the course of further development (Fig. 1). These binding sites exhibit a very low affinity, since a 1 mM concentration of unlabelled baclofen was used to determine non-specific binding. Signal transduction via metabotropic GABAB receptors was the subject of considerable experimental interest during the past decade [9]. Ontogenetic distribution of GABAB receptors in brain tissues was studied by [ 3H]GABA radioligand binding studies [5], receptor autoradiography [19], immunoblotting and immunohistochemistry [10]. Our present measurements disclosed a different distribution of GABAB receptors in three analyzed brain regions. The number of high-affinity [ 3H](2)baclofen binding sites increased progressively from birth till PD 25 (PD 12) in cortex (thalamus) and subsequently declined to adult levels. A similar developmental pattern of GABAB receptors (maximum level at PD 14 in cortex) was previously described [19]. In contrast to cortex and thalamus, we were unable to find any saturable, high-affinity [ 3H](2)baclofen binding sites in hippocampus during the first postnatal week and further development was associated only with a minor invariable radioligand binding. Contrarily, it has been reported that GABAB receptor binding peaks already at PD 3 or PD 7 in CA3 and CA1 areas of hippocampus [19]. These observations might indicate unequal distribution of these receptors in different hippocampal regions. Another [ 3H]GABA receptor binding study demonstrated lower density of GABAB sites in hippocampal tissue from 14- to 17-day-old rat pups than in adult animals [5]. These discrepancies may be due to pharmacologically distinct populations of GABAB receptors exhibiting different sensitivity and binding capacity towards different GABAB ligands [4]. The different pharmacological properties of these receptor sites, in turn, may be caused by the structural heterogeneity of GABAB receptors [13]. Next we examined the influence of baclofen on AC activity in all tested brain regions. Relatively high GTP-dependent AC activity was detected already at PD 1 in cortex, thalamus as well as in hippocampus (120, 250 and 200 pmol cAMP min 21 mg 21, respectively) and it increased dramati-
cally during the early postnatal development to reach the maximum levels at PD 12 (Fig. 2). Subsequently, AC activity gradually decreased to the adult level (PD 90), which was lower than that measured at PD 1 (Fig. 2). Baclofen significantly affected AC activity, but its influence was much smaller than the general ontogeneticallyinduced alterations in AC activity (Fig. 2). The effect of baclofen exhibited a clear resemblance to characteristics of [ 3H](2)baclofen binding. Baclofen at a 10 mM concentration exerted significant inhibition of GTP-stimulated AC activity in cortex at PD 12 and this inhibition was further increased with maturation – no effect at PD 1 and 7, inhibition by 24, 29, 37 and 46% at PD 12, 18, 25 and 90, respectively. Similar reduction of AC activity was observed in thalamus. Contrarily, GTP-dependent AC activity in hippocampal membranes prepared from 12- and 18-day-old rats was markedly enhanced by baclofen (Fig. 2). These differential modulatory effects of baclofen in the three tested brain regions were clearly dependent on the stage of ontogenetic development. Our previous study dealing with ontogenetic development of the AC signalling in rat brain provided similar results: baclofen reduced forskolin-stimulated AC in cortex and the extent of this inhibition increased with maturation. Inhibition of forskolin-stimulated AC was also observed in thalamus [8]. Contrarily, in hippocampus, baclofen did not affect forskolin-stimulated AC activity at any of the age intervals between PD 1 and PD 90. It was suggested that stimulation of 5-HT and GABAB receptors might potentiate b-adrenergic responses signalled through AC in hippocampus [1] and a region-specific enhancement of basal and neurotransmitter-stimulated AC through activation of GABAB receptors has recently been described in the rat olfactory bulb [16]. These observations are consistent with the evidence indicating that Gi-linked receptors can enhance the ability of Gs to stimulate type II and IV of AC [18]. Discrete distribution of both these AC isozymes in hippocampal formation was revealed by immunohistochemical labelling [2]. In our previous study we did not observe any substantial differences in the ontogenetic expression of AC in cortex, thalamus and hippocampus
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[8]. Adult levels of AC achieved around PD 12 and 18 were maintained in adulthood. Similarly, there were no significant discontinuities in the expression of Gs and Gi/Go proteins in these brain regions. Despite that, AC activity exhibited a pronounced maximum at PD 12, which is in agreement with our current results. Taking into consideration all these facts, we think that a specific modulation of AC activity in hippocampus can not be explained only on the basis of quantitation of AC molecules and G proteins. We would rather suppose that specific organization of neurotransmission systems in hippocampus might cause differential regulation of AC in this region, which represents a crucial brain structure for memory formation. The role of cAMP in long-term potentiation has been well documented [22]. The positive coupling of GABAB receptors to AC may be explained by the co-localization of GABAB receptors, Gi/Go proteins, Gs-linked receptors, Gs and AC molecules at specific synaptic or membrane-domain sites [16], where all these molecules co-exist and interact by functionally significant protein–protein interactions. It can not be ruled out, however, that the population of GABAB receptors in hippocampus is pharmacologically distinct from that detected in the other brain regions. This notion is supported by our present finding of a low level of low-affinity [ 3H](2)baclofen binding sites in hippocampus, which is in contrast to cortex and thalamus. It can be speculated that different levels of GABAB receptors with distinct pharmacological properties might in certain conditions exercise their regulatory function towards AC differentially. This work was supported by grants from the Grant Agency of the Czech Republic (309/99/207 and 309/01/ 0255) and by the Ministry of Education of the Czech Republic (projects LN00A026 and 11300003). [1] Andrade, R., Enhancement of b-adrenergic responses by Gi-linked receptors in rat hippocampus, Neuron, 10 (1993) 83–88. [2] Baker, L.P., Nielsen, M.D., Impey, S., Hacker, B.M., Poser, S.W., Chan, M.Y. and Storm, D.R., Regulation and immunohistochemical localization of bg-stimulated adenylyl cyclases in mouse hippocampus, J. Neurosci., 19 (1999) 180–192. [3] Bowery, N.G., GABAB receptor pharmacology, Annu. Rev. Pharmacol. Toxicol., 33 (1993) 109–147. [4] Cunningham, M.D. and Enna, S.J., Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain, Brain Res., 720 (1996) 220–224. [5] Garant, D., Sperber, E. and Moshe, S., The density of GABAB binding sites in the substantia nigra is greater in rat pups than in adults, Eur. J. Pharmacol., 214 (1992) 75–78. [6] Hill, D.R., GABAB receptor modulation of adenylate cyclase activity in rat brain slices, Br. J. Pharmacol., 84 (1985) 249– 257. [7] Hill, D.R. and Bowery, N.G., [ 3H]baclofen and [ 3H]GABA
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