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Keywords:

  • desensitization;
  • down-regulation;
  • metabotropic glutamate receptor;
  • pregnancy;
  • rat brain

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Pregnant rats were treated throughout the gestational period with either caffeine or theophylline, and its effect on the metabotropic glutamate receptor (mGluRs) signal transduction pathway was studied in both maternal and fetal brain. In maternal brain, radioligand binding assays showed that chronic treatment with methylxanthines caused a significant decrease in the total number of mGluRs. This decrease was accompanied by an increase in receptor affinity. Immunodetection showed that mGluR1a and phospholipase C β1 (PLCβ1) were significantly decreased in response to chronic methylxanthine treatment, whereas αGq/11 was not affected. A loss was also detected of PLC stimulation mediated by (S)-3,5-dihydroxyphenylglycine (DHPG), a selective Group I mGluR agonist, suggesting desensitization of the mGluR/PLC pathway. In fetal brain, a loss in total mGluRs was observed in fetuses from mothers treated with caffeine or theophylline, without variation in receptor affinity. A decrease in mGluR1a, αGq/11 and PLCβ1 levels was also observed in response to treatment. However, changes detected in this immature tissue were not associated with variations in PLC activity. These results suggest that chronic caffeine or theophylline treatment down-regulates several mGluR/PLC transduction pathway components in both maternal and fetal brain, causing a loss of receptor responsiveness only in maternal brain.

Abbreviations used
A1R

adenosine A1 receptor

AMPA

α-amino-3-hydroxy-5-methyl-isoxazole-4 propionic acid

DHPG

dihydroxyphenylglycine

GD

gestational day

GPCR

G-protein-coupled receptor

l-Glu

l-glutamic acid

mGluR

metabotropic glutamate receptor

NMDA

N-methyl-d-aspartic acid

PIP2

phosphatidylinositol 4,5-bisphosphate

PLC

phospholipase C

TBHA

dl-threo-β-hydroxyaspartic acid

Caffeine, a plant alkaloid included in the class of compounds known as methylxanthines, is the most widely used central nervous system (CNS) stimulant in the world. At doses achieved in normal human consumption, the only effect mediated by caffeine seems to be antagonism of adenosine (Fredholm et al. 1999).

Adenosine is able to modulate synaptic transmission, belonging therefore to the group of neuromodulators. The modulatory action exerted by adenosine is basically carried out at the presynaptic level, where it influences the release of neurotransmitters, and at the postsynaptic level, where it regulates postsynaptic responsiveness (Cunha 2001; Ribeiro et al. 2002). All these actions are mediated by a group of G-protein-coupled receptors (GPCRs), termed adenosine receptors, which have been classified into four subtypes, A1, A2A, A2B and A3, on the basis of pharmacological profiles, molecular cloning and preferred intracellular signalling pathway (Linden 2001). The activation of adenosine A1 receptors (A1Rs) inhibits the release of several neurotransmitters, although the most potent inhibitory effects take place on glutamate release, where synaptic transmission can be blocked in response to adenosine (Dunwiddie and Masino 2001).

Glutamate is an excitatory neurotransmitter that acts on both ionotropic and metabotropic glutamate receptors (mGluRs). mGluRs belong to the GPCR superfamily and have been classified into three groups based on sequence homology, pharmacological profiles and effector systems. Group I (mGluR1 and mGluR5) is coupled to a stimulatory Gq/11 protein and phospholipase C (PLC) activation. Group II (mGluR2 and mGluR3) and Group III (mGluR4, mGluR6, mGluR7 and mGluR8) are both coupled, in an inhibitory way, to adenylyl cyclase through a Gi/o protein (for reviews see Hermans and Challis 2001; Pin and Acher 2002; Pin et al. 2003).

Like other GPCRs, persistent activation of mGluRs often results in a loss of receptor responsiveness (Bohm et al. 1997; Ferguson 2001). This phenomenon, termed desensitization, has been described in different systems, such as C6 glioma cells, primary neuronal cultures, hippocampal slices, synaptosomes and astrocytes (De Blasi et al. 2001; Albasanz et al. 2002), and it is a consequence of the distinct mechanism that can act at the level of the three components of the transduction pathway: receptor, heterotrimeric G protein and effector system.

We previously determined that caffeine or theophylline chronically administered to pregnant rats (1 g/L in the drinking water) during gestation causes the down-regulation of adenosine A1 receptors in the brains of both mothers and fetuses, related to increased endogenous adenosine levels (León et al. 2002), and inhibits the function of A1 receptors in the maternal brain (León et al. 2005). Adenosine acting through A1 receptors inhibits glutamate release. Therefore, the aim of the present work was to study the effects of long-term exposure to the same doses of caffeine or theophylline during the gestational period on the mGluR/PLC pathway in both maternal and fetal brain. The results show that chronic caffeine or theophylline intake causes the down-regulation of several components of the mGluR/PLC pathway in both maternal and fetal rat brain, although only in maternal tissue were these changes associated with mGluR desensitization.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

l-[3H]Glutamic acid (48.1 Ci/mm) and phosphatidylinositol 4,5-bisphosphate (myo-inositol-2-3H(N)) ([3H]PIP2) (8 Ci/mm) were obtained from Dupont-NEN (Boston, MA, USA). l-Glutamate, N-methyl-d-aspartic acid (NMDA), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), (S)-3,5-dihydroxyphenylglycine (DHPG), kainate and dl-threo-β-hydroxyaspartic acid (TBHA) were from Tocris (London, UK). Phosphatidylinositol 4,5-bisphosphate (tri-ammonium salt) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Anti-PLCβ1 monoclonal antibody and anti-mGlu1a receptor polyclonal antibody were from Upstate (Reactiva, Madrid, Spain). Anti-αGq/11 protein was from Dupont NEN. All other reagents were of analytical grade.

Animals

Wistar pregnant rats, kept on a 12 h light/12 h dark cycle (lights on at 07.00 hours) and with free access to food and drinking water, were treated with 1 g/L caffeine (n = 7) or theophylline (n = 7) in the drinking water from gestational day 2 (GD 2) onwards throughout the gestational period. This high methylxanthine concentration was previously used by other authors using the same administration method (Bona et al. 1995; Johansson et al. 1997; Svenningsson et al. 1999; Da Silva et al. 2003). Also, our group has previously noted that 1 g/L caffeine or theophylline modulates the adenosine A1 receptor/adenylyl cyclase pathway in both maternal and fetal brains (León et al. 2002, 2005). The day when sperm was observed in the vaginal smear was designated day 1 of pregnancy. Control pregnant rats (n = 7) received drug-free tap water. At the end of this period (in the morning of GD 23), rats were killed by cervical dislocation and fetuses were surgically delivered. Maternal and fetal brains were then removed, frozen in liquid N2 and stored at −70°C until experiments were performed. All experiments were carried out in accordance with the Declaration of Helsinki.

Plasma membranes isolation

Brain plasma membranes from mothers and full-term fetuses were isolated as described by Kessler et al. (1989) with some modifications. Brains were homogenized in 20 volumes of isolation buffer (50 mm Tris-HCl, pH 7.4, containing 10 mm MgCl2 and protease inhibitors) in a Dounce homogenizer. After homogenization, brain preparations were centrifuged for 5 min at 1000 g in a Beckman JA 21 centrifuge. The supernatant fluid was centrifuged for 20 min at 27 000 g and the pellet finally resuspended in isolation buffer. Protein concentration was measured by the Lowry method.

Metabotropic glutamate receptor binding to plasma membranes

l-[3H]Glutamate binding assays in rat brain membranes were performed as described previously (Albasanz et al. 2002). Briefly, membranes were treated with 0.04% Triton X-100 to facilitate the removal of endogenous glutamate (Compton et al. 1990). To determine mGluR binding, 60–100 µg protein were incubated for 60 min at 25°C in the presence of 100 µm AMPA, 100 µm kainate and 100 µm NMDA, in order to block ionotropic glutamate receptor binding. Saturation assays were carried out at different l-[3H]glutamate concentrations (100–1200 nm), using l-glutamic acid (L-Glu) at a concentration 104 times that of the radioligand, in order to obtain non-specific binding. Competition curves were carried out using 250 nm l-[3H]glutamate and different concentrations (10 nm−10 mm) of L-Glu. All assays were performed in the presence of 10 µm THBA, a l-glutamate uptake inhibitor (Kimelberg et al. 1989).

Immunodetection of mGluR1a, αGq/11 and phospholipase C β1 isoform

Protein (50 µg) was subjected to 7.5% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Western blotting was performed as described earlier (Martín et al. 1998). Immunodetection was carried out by incubating the nitrocellulose membranes with specific polyclonal antibodies (anti-mGlu1a and anti-αGq/11) diluted 1 : 1000 and isoenzyme-specific monoclonal antibody (anti-PLCβ1) diluted 1 : 400. After washing, blots were incubated with horseradish peroxidase-coupled goat anti-rabbit or anti-mouse IgG diluted 1 : 3000. Antigen was visualized using the ECL chemiluminescence detection kit from Amersham (Madrid, Spain), and specific bands were quantified by scanning densitometry in a GS-690 imaging densitometer using MultiAnalyst 1.0 software from Bio-Rad (Madrid, Spain).

RT-PCR analysis

Total RNA was isolated by guanidium thiocyanate/phenol/chloroform extraction following the method of Chomczynski and Sacchi (1987). RT-PCR assays were performed as described by Vendite et al. (1998) using the primers 5′-AAATCTACAGCAATGCTGGCGA-3′ and 5′-CTTCGATGACTTCATCTCTGT-3′ for mGluR1, 5′-CGGCAAGTCTGTGTCATGGT-3′ and 5′-CAGGGTGGAAGAGCTTTGTC-3′ for mGluR1a, 5′-GAGAACCGAATGGAGGAGAGCAA-3′ and 5′-GTCCACGAACATCTTCAGGATGAA-3′ for αGq/11 and 5′-TTTTCGGCAGACCGGAAGCGA-3′ and 5′-TGCTGTTGGGCTCGTACTTCT-3′ for PLCβ1. PCR products were analysed by electrophoresis in 2% agarose gels and stained with ethidium bromide. The expected PCR product sizes for mGluR1, mGluR1a, αGq/11 and PLCβ1 were 206, 930, 212 and 315 bp, respectively. In all cases, amplification of a fragment corresponding to the β-actin sequence was carried out in parallel, using the same cDNA samples, in order to correct possible variations in the amount of cDNA used for the process. The primers used for β-actin were 5′-GGTATGGAATCCTGTCGCATCCATGAAA-3′ and 5′-GTGTAAAACGCAGCTCAGTAACAGTCCG-3′. The size of the PCR product for β-actin was 320 bp. Bands corresponding to PCR products were quantified by densitometry in a Bio-Rad GS-690 densitometer using MultiAnalyst 1.0 software.

Phospholipase C assay

Phospholipase C activity in plasma membranes was assayed in the presence of exogenous [3H]PIP2 as described by Tiger et al. (1990). [3H]PIP2 was dried under an N2 stream, dissolved in 2 mm sodium deoxycholate and 50 mm Tris-HCl pH 6.5, and sonicated using an Ultrasonic Processor UP 200 S. The phospholipase C assay was carried out for 10 min at 37°C, incubating [3H]PIP2 (17 000 d.p.m.) with or without 20 µg plasma membrane protein in 100 µL buffer (100 mm NaCl, 1 mm sodium deoxycholate, 1 mm EGTA, 25 µm CaCl2, 40 mm diCl and 50 mm Tris-HCl pH 6.8). Functionality of the transduction pathway was determined by stimulation with 100 µm GTPγS Guanosine-S1-O-(3-thio-triphosphate), a non-hydrolysable GTP analogue that activates phospholipase C through G protein, and with 100 µm DHPG, specific group I mGluR agonists.

The incubation was terminated by the addition of 360 µL chloroform/methanol/HCl (1 : 2 : 0.2 v/v) and placing the tubes on ice. After addition of 120 µL 2 m KCl and 160 µL chloroform, the tubes were centrifuged for 5 min at 3500 g. The upper aqueous phase (250 µL) containing [3H]inositol phosphates was mixed with 3.5 mL scintillation liquid.

Statistical and data analysis

Statistical analysis of the data was performed using Student's t-test. Differences between mean values were considered statistically significant at p < 0.05. Saturation (Bmax, KD) binding curves were analysed by Scatchard, and non-linear regression analysis of binding data with the GraphPad Prism 3.03 program (GraphPad Software, San Diego, CA, USA). The IC50 values for l-[3H]glutamate were converted to Ki values according to the Cheng-Prusoff equation Ki = IC50/(1 + [L]/KD) (Cheng and Prusoff 1973) using the KD values shown in Table 1 for the different treatments.

Table 1.  Kinetic parameters of l-[3H]glutamate binding to plasma membranes from maternal and fetal brain. Data represent Bmax and KD from maternal and fetal membranes determined by Scatchard analysis of binding data shown in Figs 1 and 2. Data are means ± SEM from at least four independent experiments performed with different plasma membrane isolations
 MothersFetuses
Bmax (pm/mg protein)KD (nm)Bmax (pm/mg protein)KD (nm)
  • ***

    p < 0.001,

  • **

    p < 0.01 and

  • *

    p < 0.05 significantly different from control values.

Control14.6 ± 2.5534.3 ± 89.815.3 ± 2.6810.6 ± 111.3
Caffeine4.5 ± 0.4***461.8 ± 80.2*11.2 ± 3.3*695.9 ± 106.6
Theophylline4.1 ± 1.7***309.6 ± 118.4**13.1 ± 1.0*802.5 ± 158.9

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Water intake was measured daily in all groups of rats (control, caffeine- or theophylline-treated). Caffeine or theophylline consumption was estimated from the loss of water from the drinking bottles. Caffeine and theophylline intake was 83.2 ± 5.3 and 83.8 ± 2.2 mg/kg day, respectively, whereas the tap water intake of the control group was 84.4 ± 5.5 mL/kg day. Caffeine or theophylline do not significantly altered the weight of mothers at the end of the gestational period (data not shown). The average daily caffeine or theophylline consumption was in a dose range previously reported (Johansson et al. 1993, 1997; Svenningsson et al. 1999).

Chronic caffeine or theophylline consumption did not affect the number of live fetuses at birth. The average litter size obtained at the end of pregnancy was 11 ± 1.3 (7), 10 ± 1.0 (7) and 10 ± 1.5 (7) from control, caffeine- or theophylline-treated rats, respectively (number of mothers analysed in parentheses). Chronic consumption of neither caffeine nor theophylline modified the body weight of fetuses at birth (the mean values were 5.8 ± 0.3 and 5.5 ± 0.2 g in the caffeine and theophylline-treated groups vs. 5.6 ± 0.4 g in the control group). Similar conclusions were reached when we weighed the total fetal brain in the three groups of animals. Finally, no significant incidence of congenital malformations was found in fetuses from mothers treated with caffeine or theophylline compared to controls.

Effect of chronic treatment with caffeine or theophylline on mGluRs in both maternal and fetal brain

The effect of chronic treatment with methylxanthines on total mGluRs was analysed in plasma membranes, obtained from maternal and fetal brain, by saturation binding assays using l-[3H]glutamate as radioligand. As can be seen in Fig. 1 and Table 1, chronic caffeine or theophylline exposure produced a significant decrease in number of total mGluRs. This diminution was of the same order in caffeine- (70%) and theophylline-treated (72%) animals, indicating that both antagonists caused the same effect at the receptor level. This loss of mGluRs was also accompanied by a decrease in KD value, suggesting an increase in receptor affinity. The same analysis performed on fetal brain showed a similar significant decrease in total mGluRs in caffeine- (27%) and theophylline-treated (15%) fetuses (Fig. 2 and Table 1). However, these changes were not associated with variations in receptor affinity.

image

Figure 1. Saturation curves of l-[3H]glutamate binding to plasma membranes from maternal brain. Aliquots of 50 µg of plasma membranes from control, caffeine- or theophylline-treated pregnant rat brain were incubated with different concentrations of l-[3H]glutamate, as described in Materials and methods, after pretreatment with 0.04% Triton X-100, in order to remove endogenous glutamate from the samples. Total receptor number (Bmax) and receptor affinity (KD) were determined by Scatchard analysis of saturation curves and these values are shown in Table 1. Data are mean ± SEM of five to seven experiments performed using different plasma membrane isolations.

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image

Figure 2. Saturation curves of l-[3H]glutamate binding to plasma membranes from fetal brain. Binding of l-[3H]glutamate to fetal brain plasma membranes from control and caffeine- or theophylline-treated rats was performed as described in Materials and methods. Kinetic parameters are shown in Table 1 and were determined by Scatchard analysis. Data are mean ± SEM of at least five experiments performed using different plasma membranes isolations.

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The higher affinity found in maternal brain in response to chronic caffeine or theophylline treatment was corroborated by competition curves of l-glutamate. As demonstrated in Fig. 3a and Table 2, the l-glutamate competition curve in treated pregnant rats showed lower IC50 and Ki values than those in control rats. On the other hand, no significant differences in corresponding IC50 and Ki values were observed in fetal brain, in agreement with the absence of KD variations detected in the saturation curves (Fig. 3b).

image

Figure 3. l-Glutamate competition curves of l-[3H]glutamate binding to plasma membranes from maternal and fetal brain. Plasma membranes from maternal (a) and fetal (b) brain were incubated with 250 nm l-[3H]glutamate, as described in Materials and methods, without or with increasing concentrations of l-glutamate in a concentration range from 10−8 to 10−2m. Data are mean ± SEM of three experiments performed using different plasma membranes isolations.

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Table 2. l-Glutamate competition curves from maternal and fetal brain plasma membranes. Data represent IC50 values determined by non-linear regression analysis in both maternal and fetal plasma membranes and the corresponding Ki values calculated with the Cheng-Prusoff equation. Data are means ± SEM from at least three experiments performed with different plasma membrane isolations
 MothersFetuses
IC50 (nm)Ki (nm)IC50 (nm)Ki (nm)
  • **

    p < 0.01 significantly different from control.

Control875.1 ± 148.2596.1 ± 100.9157.4 ± 32.2140.1 ± 28.6
Caffeine490.5 ± 43.5**318.1 ± 28.1**182.4 ± 44.6159.3 ± 38.9
Theophylline383.4 ± 104.4**211.9 ± 57.8**125.2 ± 42.2111.3 ± 37.5

To analyse changes observed in mGluRs further, we determined the steady-state level of mGluR1a subtype in both maternal and fetal brain by immunoblotting assays using specific anti-mGluR1a antibody. As shown in Fig. 4, the immunodetection of this subtype was significantly lower in brain plasma membranes from caffeine- (86 ± 4%) or theophylline-treated (76 ± 6%) mothers. With respect to fetal brain, mGluR1a immunodetection was also lower in the groups treated with caffeine (63 ± 11%) or theophylline (58 ± 5%).

image

Figure 4. Immunoblotting analysis of mGluR1a in plasma membranes from maternal and fetal brain. Aliquots of 50 µg of plasma membranes from control and caffeine- or theophylline-treated rat brain were subjected to SDS–PAGE, transferred electrophoretically to nitrocellulose membrane and incubated with a specific polyclonal antibody (anti-mGlu1a), as described in Materials and methods. Data, expressed as percentage of control values, are mean ± SEM of three to four experiments performed using different plasma membrane isolations. Inset shows representative western blot; **p < 0.01 and *p < 0.05 significantly different from control.

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To determine whether caffeine or theophylline intake affects mRNA coding mGluR1a, we performed RT-PCR following total RNA isolation from control and treated brain, using specific primers corresponding to mGluR1a and β-actin. As can be seen in Fig. 5, chronic caffeine or theophylline treatment during the whole gestational period did not produce any significant change in mGluR1a mRNA level in either maternal or fetal brain. We also analysed mGluR1 mRNA but no significant differences were detected in any case (data not shown). These results suggest that a post-transcriptional mechanism is responsible for the down-regulation of mGluR1a.

image

Figure 5. Effect of chronic caffeine or theophylline treatment on mGluR1a expression detected by RT-PCR. After RNA isolation from maternal and fetal brain, RT-PCR assays were performed using specific oligonucleotides to mGluR1a as described in Materials and methods. The histograms show the ratio between amplification of mGluR1a and amplification of β-actin using the same RNA samples. Data are mean ± SEM of three experiments performed with different RNA isolations. Inset shows a representative experiment of the amplification of mRNA encoding mGluR1a protein.

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Effect of chronic treatment with caffeine or theophylline on αGq/11 protein in both maternal and fetal brain

It has been shown that chronic agonist stimulation can produce a down-regulation of heterotrimeric G-protein (Milligan 1993). In order to investigate whether chronic caffeine or theophylline intake affected αGq/11 level, we performed immunoblotting assays using a specific antibody against this protein. The analysis of the band specifically recognized by the antibody in maternal brain did not reveal significant differences between membranes from control and treated animals. However, chronic caffeine or theophylline treatment produced a slight but significant decrease of 12% and 15%, respectively, in the immunolabelling of αGq/11 in membranes from fetal brain (Fig. 6).

image

Figure 6. Immunoblotting analysis of αGq/11 in plasma membranes from maternal and fetal brain. Plasma membranes (50 µg) from control and caffeine- or theophylline-treated rat brain were subjected to SDS/PAGE, transferred electrophoretically to nitrocellulose membrane and probed with anti-αGq/11 antisera, as described in Materials and methods. Data, expressed as percentage of control values, are mean ± SEM of four to five experiments performed using different plasma membrane isolations. Inset shows αGq/11 bands corresponding to a representative experiment; **p < 0.01 significantly different from control.

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The level of mRNA coding αGq/11 was studied by RT-PCR. Analysis of the specific band showed that chronic caffeine or theophylline treatment did not modify the mRNA level in either mothers or fetuses (Fig. 7). The absence of modulation agrees well with the lack of variation in the corresponding protein level in maternal brain. However, the absence of changes in αGq/11 mRNA in fetal brain contrasted with the slight but significant decrease observed, with high reproducibility, in this protein by immunoblotting. This suggests the involvement of post-transcriptional modifications in the slight down-regulation of αGq/11 protein detected in the immature tissue.

image

Figure 7. RT-PCR analysis of mRNA encoding αGq/11 proteins in maternal and fetal brain. RT-PCR assays were performed using RNA isolated from control and caffeine- or theophylline-treated rat brain and oligonucleotides specific to αGq/11, as described in Materials and methods. Results are expressed as the ratio between amplification of αGq/11 and amplification of β-actin mRNAs. Data are mean ± SEM of three experiments performed with different RNA isolations. Inset shows the amplification of mRNA encoding αGq/11 protein of a representative experiment.

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Effect of chronic treatment with caffeine or theophylline on phospholipase C in both maternal and fetal brain

After investigating the components mGluR1a and αGq/11 of the mGluR/PLC transduction pathway, we analysed the effect of chronic caffeine or theophylline intake on phosphoinositide-specific PLC, the main effector system coupled to Group I mGluRs. First, we analysed the PLCβ1 isoform by western blotting using an antibody that specifically recognizes this protein and that migrates as 150 kDa peptides on SDS polyacrylamide gels. Analysis of the immunoblots showed that chronic treatment of pregnant rats with caffeine or theophylline significantly reduced the intensity of PLCβ1 immunoreactivity by 27% and 26%, respectively, in maternal brain, and by 18% and 23% in fetal brain membranes (Fig. 8). The reduction in PLCβ1 content in maternal brain contrasted with the absence of variations in the steady-state level of the PLCβ1 mRNA transcript, suggesting that a post-transcriptional mechanism may be involved (Fig. 9).

image

Figure 8. Immunoblotting analysis of PLCβ1 in plasma membranes from maternal and fetal brain. Plasma membranes (50 µg) from control and caffeine- or theophylline-treated rat brain were subjected to SDS–PAGE, transferred electrophoretically to nitrocellulose membrane and probed with anti-PLCβ1 antisera, as described in Materials and methods. Data, expressed as percentage of control values, are mean ± SEM of three to four experiments performed using different plasma membrane isolations. Inset shows PLCβ1 bands corresponding to a representative experiment; **p < 0.01 significantly different from control value.

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image

Figure 9. Analysis of the expression of the PLCβ1 in maternal and fetal rat brain by RT-PCR. RT-PCR assays were performed using 5 µg total RNA isolated from control and caffeine- or theophylline-treated rat brain, and oligonucleotides specific to PLCβ1, as described in Materials and methods. The histograms show the ratio between amplification of PLCβ1 and amplification of β-actin mRNAs using the same RNA samples. Data are mean ± SEM of three experiments performed with different RNA isolations. Inset shows the amplification of mRNA encoding PLCβ1 protein of a representative experiment.

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Secondly, we evaluated basal PLC activity in membranes from the mothers' brains using [3H]PIP2 as an exogenous substrate (Fig. 10). The results indicated that basal PLC activity in control group (392 ± 41 pm/mg/min) was not significantly different from the caffeine (383 ± 29 pm/mg/min) or theophylline (446 ± 46 pm/mg/min) groups. The activation of PLC through G-protein was also studied using GTPγS, a non-hydrolysable GTP analogue. A percentage of stimulation of 100 µm GTPγS above the control values was significantly lower in brain membranes from caffeine- or theophylline-treated rats (116 ± 4% and 119 ± 2%, respectively) than control rats (139 ± 8%). The loss of GTPγS-mediated PLC stimulation in caffeine- or theophylline-treated groups contrasted with the lack of variation in αGq/11 protein level shown above, which seems to indicate that chronic treatment decreased the affinity of GTPγS for PLC.

image

Figure 10. Effect of chronic caffeine or theophylline treatment on phospholipase C activity in plasma membranes from maternal brain. PLC activity was measured as described in Materials and methods using [3H]PIP2 as exogenous substrate. Plasma membranes (20 µg) were incubated in the presence of 100 µm GTPγS, 100 µm DHPG or 100 µm GTPγS plus 100 µm DHPG. Data are mean ± SEM of three to four experiments performed in duplicate using different plasma membrane isolations; ***p < 0.001 and **p < 0.01 significantly different from control values.

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Thirdly, we assayed the functional consequences of the mGluR1a down-regulation observed in maternal brain membranes from rats treated with caffeine or theophylline. The stimulation of PLC mediated by DHPG, a specific Group I mGluR agonist, was significantly lower in caffeine- (122 ± 8%) and theophylline-treated (123 ± 1%) groups than in the control group (144 ± 3%). This suggests that chronic methylxanthine treatment causes desensitization in the mGluR/PLC transduction pathway in maternal brain. In agreement with the results obtained with DHPG and GTPγS, the co-stimulation of PLC with both compounds was significantly reduced in caffeine- (143 ± 5%) and theophylline-treated (150 ± 2%) groups compared with controls (177 ± 7%).

PLC activity was also assayed in fetal brain membranes (Fig. 11), where neither basal activity nor GTPγS-, DHPG- or DHPG plus GTPγS-mediated PLC stimulation changed in response to chronic caffeine or theophylline treatments.

image

Figure 11. Effect of chronic caffeine or theophylline treatment on phospholipase C activity in plasma membranes from fetal brain. PLC activity was measured as described in Materials and methods using [3H]PIP2 as exogenous substrate. Plasma membranes (20 µg) were incubated in the presence of 100 µm GTPγS, 100 µm DHPG or 100 µm GTPγS plus 100 µm DHPG. Data are mean ± SEM of three to four experiments performed in duplicate using different plasma membrane isolations.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

At doses reached by normal caffeine consumption in humans, this methylxanthine blocks A1 and A2A receptors (Daly and Fredholm 1998). Adenosine through its binding to A1R can interact with several neurotransmitters such as GABA (Akhondzadeh and Stone 1994), ATP (Gerwins and Fredholm 1992), histamine (Dickenson and Hill 1994), dopamine (Ferréet al. 1997) and glutamate (Vazquez et al. 1995). Thus, chronic caffeine or theophylline exposure can affect transduction pathways other than adenosine receptors. In the present work, we show that chronic gestational treatment with caffeine or theophylline down-regulates components of the mGluR/PLC transduction pathway in both maternal and fetal brain, and that these changes are associated with a loss of receptor responsiveness in mature brain.

The significant reduction in the mGluR population from maternal brain was accompanied by an increase in receptor affinity, suggesting the possible existence of a compensatory mechanism to counteract the large decrease in mGluRs. A corresponding analysis in fetal brain also indicated a loss of mGluRs in fetuses from mothers treated with caffeine or theophylline, without alterations in the binding affinity of these receptors. Our group previously recorded that caffeine or theophylline consumption causes the down-regulation of adenosine A1 receptors in both maternal and fetal brain (León et al. 2002), and inhibits A1 receptor function in the maternal rat brain (León et al. 2005). The down-regulation of A1R has been associated with a loss of receptor functionality in several tissues. Thus, it has been reported to attenuate the inhibition of lipolysis and adenylyl cyclase activity mediated by A1R in non-neural systems such as adipocytes and cardiac myocytes (Green 1987; Liang and Donovan 1990). On the other hand, the decrease of A1R in cerebellar granule cells and in rat brain membranes has also been linked to a diminution in A1R-mediated inhibition of AC (Hettinger-Smith et al. 1996; Ruiz et al. 1996; Vendite et al. 1998). Therefore, it is possible that the loss of A1R detected after chronic gestational treatment with caffeine and theophylline (León et al. 2002) in maternal and fetal brain, and the decreased A1R function in the maternal brain (León et al. 2005), can modify the A1-mediated inhibition of glutamate release (Dunwiddie and Masino 2001). Supporting this hypothesis, prolonged treatment with adenosine A1R agonist in hippocampal neurones decreased the presynaptic inhibition of endogenous glutamate release, probably associated with the down-regulation of A1R (Ambrosio et al. 1997; Wetherington and Lambert 2002). Additionally, acute intraperitoneal administration of caffeine in rats produced an increase in extracellular levels of glutamate in the nucleus accumbens (Solinas et al. 2002). In any case, the antagonism of adenosine A1R produced by short-term exposure to caffeine as well as the down-regulation of this receptor after chronic caffeine or theophylline treatment could result in a loss of A1R responsiveness, which at presynaptic level would produce an increase in glutamate release that, in turn, would evoke the down-regulation of mGluRs. In this sense, the down-regulation of mGluR reported here could be a response to receptor over-stimulation produced by excessive glutamate release. In agreement with our results down-regulation of mGluR1 in C6 glioma cells (Albasanz et al. 2002) and mGluR5 in astrocytes (Balazs et al. 1997) after prolonged treatment with mGluR agonists has been demonstrated. Similar results showing a loss of mGluR1a on the membrane surface have also been reported in HEK 293 cells following agonist exposure (Dale et al. 2001; Mundell et al. 2001, 2003).

The immunodetection of mGluR1a carried out here has shown that this receptor subtype contributes to the mGluR down-regulation detected by kinetic experiments in both maternal and fetal brain. However, after comparing the results obtained in radioligand binding and western blotting assays, we cannot exclude the regulation of other mGluR subtypes after treatment. Additionally, the lack of variation in mGluR1 and the splicing variant mGluR1a mRNA level in both tissues points to a post-transcriptional mechanism being responsible for the down-regulation. In support of this hypothesis, endocytosis of mGluRs through clathrin-coated vesicles following agonist exposure has been demonstrated (Mundell et al. 2001; Albasanz et al. 2002).

Loss of receptor responsiveness is a phenomenon widely described upon agonist stimulation. Although the mechanisms involved in receptor desensitization can operate at the level of the three components of the transduction pathway, most studies have focused on the events that take place at receptor level. The down-regulation of mGluR detected in maternal brain following chronic caffeine or theophylline treatment was associated with a loss of mGluR/PLC functionality, in agreement with the decreased immunodetection of mGluR1a. This result agrees well with the agonist-induced desensitization of Group I mGluR detected in different systems, such as synaptosomes, hippocampal slices, astrocytes, neuronal cultures and recombinant systems (for review see De Blasi et al. 2001; Hermans and Challis 2001). However, the decrease in mGluR and mGluR1a observed in fetal brain was not accompanied by reduced mGluR/PLC responsiveness. This lack of effect could be related to the different expression of the PLCβ1 isoform in fetal brain at term, that was only 44 ± 7% of the maternal level, and the lower basal PLC activity detected in this young tissue (50% of maternal activity). Accordingly, immunodetection of this enzyme at GD 19 in rat cerebral cortex was virtually absent compared with the level observed at 4 weeks postnatally (Shimohama et al. 1998).

Apart from receptor protein, the heterotrimeric G-protein and the effector system may also be down-regulated in response to chronic agonist exposure contributing to the attenuation of receptor signalling (Bohm et al. 1997). Consistent with this idea, the persistent activation of transfected human M3 or M1 muscarinic acetylcholine receptors has been reported to produce the down-regulation of αGq/11 in CHO cells (Mullaney and Milligan 1993; van de Westerlo et al. 1995). However, in the present work, the immunoblot analysis of αGq/11 in maternal brain did not reveal significant differences between the control and the caffeine or theophylline groups. This absence of variation in αGq/11 level contrasted with the slight, but significant, down-regulation of this protein detected in fetal brain. This apparent discrepancy could be related to the different levels of αGq/11 detected in both maternal and fetal brain. Thus, the immunodetection of this protein is 28% lower in maternal brain than fetal brain, in accordance with the ontogenic studies of heterotrimeric G-proteins carried out in rat brain where the αGq/11 levels detected at postnatal day 90 corresponded to 50% of the levels observed at birth (Ihnatovych et al. 2002). In this way, it is possible that the effect of chronic agonist activation on heterotrimeric G-protein level depends on stoichiometry, being more evident in fetal brain where the αGq/11 level is higher with respect to maternal brain (Hettinger et al. 1998). On the other hand, the down-regulation of αGq/11 in fetal brain was not accompanied by a loss of GTPγS mediated-PLC stimulation, suggesting that the availability of αGq/11 does not limit the rate of phosphoinositide hydrolysis in spite of the loss of αGq/11 following chronic methylxanthine treatment.

With respect to the effector system, the results obtained in the present study agree with the hypothesis that treatment affects mGluR/PLC signalling by decreasing the presynaptic inhibition of glutamate release mediated by A1R. Thus, the decrease in PLCβ1 detected after treatment in both maternal and fetal brain agrees well with one of the mechanisms used by the cell to protect against the continuous activation by agonist, i.e. the down-regulation of the effector system (Bohm et al. 1997). Our results agree with the down-regulation of PLCβ1 detected in SH-SY5Y cells following the chronic activation of muscarinic receptor (Sorensen et al. 1998). However, basal PLC activity did not undergo any significant variation in either tissues. A possible explanation could be the presence of other important PLC isoenzymes, such as PLCγ1 and PLCδ1, in the cerebral cortex at embryonic day 19 and at 48 postnatal weeks (Shimohama et al. 1998). Thus, it is possible that PLCγ1 and PLCδ1 basal activities contribute to the [3H]PIP2 hydrolysis detected under our basal conditions, occluding the down-regulation of the PLCβ1 isoform.

On the other hand, the absence of variation in the mRNA level of PLCβ1 in both maternal and fetal brain after treatment, in spite of the decrease observed by immunoblotting, suggests the involvement of a post-transcriptional regulatory mechanism, such as internalization, in the down-regulation of the effector system. In support of this hypothesis our laboratory has previously reported the presence of PLCβ1 coupled to mGluRs in clathrin-coated vesicles (Martín et al. 1991, 1995).

In conclusion, the present work demonstrates that chronic caffeine or theophylline treatment during the gestational period produces a desensitization of mGluR/PLC signalling in the maternal brain. This loss of receptor responsiveness may be explained by the down-regulation of mGluR1a and PLCβ1 detected at the end of the treatment. On the other hand, although mGluR1a, αGq/11 and PLCβ1 were down-regulated in fetal brain in response to chronic caffeine or theophylline exposure, no changes in mGluR/PLC responsiveness were detected, possibly because of the immaturity of mGluR/PLC signalling at birth. These results confirm the existence of cross-talk between transduction pathways mediated by adenosine and glutamate, suggesting that caffeine and theophylline consumption during gestation should be restricted.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by grants G03/167 and C03/06 from Instituto de Salud ‘Carlos III’, grant PAI02-003 from the Consejería de Ciencia y Tecnología of JCCM, and BFI2002-00277 from DGES (MEC). This article is dedicated to the memory of the innocent victims of the terrorism attack on March 11, 2004 in Madrid, Spain.

References

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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