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Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions
Neurochemistry International 46 (2005) 347–356 www.elsevier.com/locate/neuint
Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions P. de Gortaria,*, F. Romerob, M. Cisnerosb, P. Joseph-Bravob a
Department Neurociencias, Instituto Nacional de Psiquiatrı´a, Ramo´n de la Fuente Mun˜iz, Calzada Me´xico-Xochimilco 101, Col. San Lorenzo Huipulco, C.P. 14370, Me´xico b Department Gene´tica del Desarrollo y Fisiologı´a Molecular, Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, Me´xico Received in revised form 31 March 2004; accepted 12 April 2004 Available online 31 December 2004
Abstract Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABAA receptors’ agonists and antagonists were tested at 1 h (ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol’s inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol’s inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons. # 2004 Elsevier Ltd. All rights reserved. Keywords: Pro-TRH mRNA; PPII regulation; NMDA; GABA
1. Introduction In the central nervous system (CNS), efficient inactivating mechanisms limit the time the neurotransmitters remain in the extracellular fluid. Most classical neurotransmitters are rapidly removed from the synaptic cleft by specific transporters (uptake) (Iversen, 1971). In the case of neuropeptides, the principal mode of inactivation, once released, is their degradation by ectopeptidases that usually have a broad * Corresponding author. Tel.: +52 56 55 28 11x114; fax: +52 56 55 99 80. E-mail address: [email protected] (P. de Gortari). 0197-0186/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2004.11.002
specificity. In general, various enzymes can degrade a given peptide; for example, opioids, cholecystokinin, somatostatin and substance P are degraded by neprilysin (NEP, EC 3.4.24.11; ‘‘enkephalinase’’), aminopeptidase N (APN, E.C. 3.4.11.2) or angiotensin converting enzyme (ACE, E.C. 3.4.15.1) (Csuhai et al., 1995; O’Cuinn et al., 1995; Turner et al., 2001). An exception is pyroglutamyl aminopeptidase II (PPII, EC 3.4.19.6) whose only known endogenous substrate is thyrotropin-releasing hormone (TRH) (Csuhai et al., 1995; O’Cuinn et al., 1995). PPII is enriched in nerve endings and has an heterogeneous localization; PPII mRNA distribution coincides with either one of TRH receptor mRNAs (Charli et al., 1998; Heuer et al., 2000). Evidence accumulates
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supporting the inactivating mechanisms as an additional point of homeostatic control; transporters and ectopeptidases can be regulated at different levels and this regulation can in turn play an active role in neurotransmission (Danbolt, 2001; Horschitz et al., 2001; Reith et al., 1997; Antczak et al., 2001; Charli et al., 1998; Konkoy and Davis, 1996; Heuer et al., 1998). In CNS, the expression and activity of NEP and APN are modulated by acute or chronic administration of neuroleptic drugs that affect dopaminergic transmission, or by chronic alcohol consumption; this regulation differentially modifies the rate of degradation of substance P, met-enkephalin or neurotensin (Konkoy and Davis, 1996; Waters et al., 1995, 1997). Activity of these enzymes differs between alcohol preferring and alcohol avoiding mice and rat lines; administration of NEP and ACE inhibitors decreases alcohol-drinking behavior in mice (Winkler et al., 1998) or rats (Grupp and Chow, 1992). Our group has provided evidence for a stringent control of PPII in the adenohypophysis, a target of TRH in its neuroendocrine role. In cultured adenohypophyseal cells, PPII expression and activity are regulated by its substrate TRH, or by drugs that alter protein kinase C (PKC), protein kinase A (PKA), calcium levels or arachidonic acid cascades (Charli et al., 1998; Baeza et al., 2001; Vargas et al., 1998, 2002a). In vivo, PPII is also modulated by TRH and hormones (Charli et al., 1998; Heuer et al., 1998; Vargas et al., 2002b). Modulation of brain PPII activity was demonstrated in a paradigm that activates TRHergic neurons in limbic areas (amygdaloid kindling) (de Gortari et al., 1995). PPII activity is elevated in hippocampus, amygdala and cortex during kindling development (stage II); while it is decreased in fully kindled rats (stage V), when TRH content and mRNA levels are increased (de Gortari et al., 1995, 1998; Knoblach and Kubek, 1997; Rosen et al., 1994). At this later stage, TRH receptor binding is also diminished (Kubek et al., 1993). TRH is widely distributed in the CNS and its administration causes several behavioral changes that support, together with in vitro experiments, its neuromodulatory role (Horita, 1998; Stocca and Nistri, 1995; Kasparov et al., 1994). TRH has been implicated in arousal (Thompson and Rosen, 2000), locomotor activity (Metcalf and Dettmar, 1981) and autonomic functions (Doong and Yang, 2003). Its administration produces antidepressive, antiepileptic or neuroprotective effects (Horita, 1998; Sattin et al., 1999), and shortens the time of narcosis induced by ethanol and barbiturates (Morzorati and Kubek, 1993; French et al., 1993); improves cognitive function in animals affected by chronic ethanol administration (Khan et al., 1993) and blocks the locomotor suppression induced by high doses of ethanol (Breese et al., 1985). An acute administration of ethanol provokes a fast and transient increase in TRH expression in hippocampus and n. accumbens, and a decrease in frontal cortex. The kinetics of the response is different, not only among regions but also to that observed for proenkephalin mRNA suggesting a specific response (de Gortari et al., 2000a).
The aim of this work was to study the effect of an acute ethanol administration on TRH extracellular inactivation measuring PPII activity and its mRNA levels in limbic regions. Since ethanol inhibits NMDA receptors (NMDAR) and activates GABAA receptors (GABAAR) (Crews et al., 1996; Khanna et al., 2002; Besheer et al., 2003) while kindling progression correlates with the increased activation of NMDAR and with decreased GABA receptors (GABAR) activity (Faingold et al., 1998; Sutula et al., 1996) we measured PPII expression and activity, as well as TRH gene expression, in rats receiving agonists or antagonists of these receptors and their possible modulation of ethanol effects.
2. Experimental procedures 2.1. Animal handling and drug administration Male Wistar rats (400–500 g) from the Institute’s colony were fed (Purina Chow) ad libitum and kept in 07:19-h light:dark cycle and controlled temperature (25 8C). The Ethics Committee and Project Commission of the Instituto Nacional de Psiquiatrı´a approved the experiments and care was taken to avoid unnecessary stress. In order to avoid changes due to circadian rhythms, injections were performed at different times in such a way that animals were sacrificed between 11–12:00 h by decapitation. 2.1.1. Protocol 1: kinetics of ethanol effect on PPII activity Rats were injected i.p. with ethanol (3 g/kg of body weight (b.w.) diluted to 25% in 0.9% saline; Baker, Me´ xico); controls were injected simultaneously with equivalent volumes of saline solution. Injections were performed at 1, 6 or 24 h before the time of sacrifice (11:00 h). Six groups of four rats were formed, one control and one experimental for each time (24 animals); two independent experiments were performed (48 animals, n = 8). PPII activity was measured in cellular membranes extracted from hypothalamus, amygdala, n. accumbens, frontal cortex, and hippocampus as described below. 2.1.2. Protocol 2: kinetics of ethanol effect on PPII mRNA levels Based on the effects on PPII activity, and with the aim to analyze if some of those changes could be due to alterations in expression levels of the PPII gene, we varied the time of ethanol injection previous to sacrifice (0.5 h for frontal cortex and 1, 2, 4 and 16 h for the other regions). Each time had its control group so that 10 groups of 4 rats were formed (2 independent experiments). Total RNA of each region (same as in protocol 1) was extracted and analyzed for PPII mRNA levels by RT-PCR as described below. 2.1.3. Protocol 3: effects of drugs altering glutamate or GABA transmission N-methyl-D-aspartate (NMDA) and its antagonist MK801, muscimol (GABAA agonist) and its antagonist
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picrotoxin, were injected i.p. either alone or 30 min before an ethanol injection. Animals injected with ethanol, muscimol or MK-801 were narcotized almost immediately and started to wake up around the time of sacrifice. Animals were sacrificed 1 h after the ethanol injection or after the single injection of the other drugs. Drugs were used at concentrations recommended in the literature (Aversano et al., 2002; Le et al., 1992; Toropainen et al., 1997; Zarrindast and Mousa-Ahmadi, 1999): N-methyl-D-aspartate (30 mg/kg b.w.); MK-801 (dizocilpine; 0.1 mg/kg b.w.); muscimol, (4 mg/kg b.w.); picrotoxin (5 mg/kg b.w.), each diluted in the same volume (5 ml) of 0.9% saline solution, (purchased from SigmaAldrich, St. Louis, MO, USA). Three experiments were performed, each with four animals per group and a control group injected with saline solution. Frontal cortex, hippocampus and n. accumbens were the brain regions dissected.
protein, 1 mM of protease inhibitors N-ethylmaleimide and bacitracin, 17 mg/ml dipeptidyl aminopeptidase (DAP IV; Sigma-Aldrich, St. Louis, MO, USA). After 5 min preincubation at 37 8C, 10 ml (400 mM final) of TRH-bnaphtylamide (TRH-b-NA) were added as substrate and tubes were further incubated for 15, 30 and 60 min at 37 8C. Reaction was stopped with 250 ml of methanol. b-NA production was detected fluorometrically.
2.2. Tissue collection and sample preparation
Frozen brain regions were homogenized in 4 M guanidine thyocianate (ICN, Aurora, OH, USA) and total RNA extracted as described (Chomczynski and Sacchi, 1987). PPII and TRH mRNA were semi-quantified by reverse transcriptase polymerase chain reaction (RT-PCR) using two different control transcripts: glyceraldehyde 3phosphate dehydrogenase (G3PDH) or cyclophilin. Analysis of proTRH or PPII mRNA levels using G3PDH mRNA as control was performed as reported (de Gortari et al., 2000a; Vargas et al., 1998), except that 15 pmol per tube of G3PDH primers and 25 cycles were used. A 257 bp fragment corresponding to nucleotides 165–422 of the cyclophilin cDNA was also amplified for 23 cycles using 50 pmol per tube of primers whose sequences were: 50 -ACA-TGC-TTGCCA-TCC-AGC-C-30 , antisense: 50 -GGG-GAG-AAAGGA-TTT-GGC-TA-30 . Ten microliters of proTRH or PPII PCR product and 5 ml of either G3PDH or cyclophilin PCR product were separated by a 2% agarose gel (Ultra-pure BioRad, Hercules, CA, USA) electrophoresis; gels were stained with ethidium bromide (1 mg/L) and measured by densitometry (Bio-Rad Fluor-S Multi-Imager System and Multianalyst program). The relative amounts of PPII or TRH mRNA were calculated as the ratio of TRH amplicon over that of control amplicon.
Brains were excised (taking care of cutting the optic nerve before removal from skull, to preserve intact the median eminence), and kept at 70 8C until dissection. Hypothalamus, amygdala, n. accumbens, frontal cortex and hippocampus were hand dissected from frozen brains following Palkovits and Brownstein (1988) coordinates. For protocols 1 and 2, the left and right regions were pooled. For protocol 3, left or right regions were stored separately. This later protocol was performed three times and analyzed as follows: (a) experiment 1: one half of each region was used to measure PPII activity and the other half, for RNA extraction to measure mRNA levels of PPII and TRH; (b) experiment 2: TRH content in one half, and RNA extraction in the other half; (c) experiment 3: one half for PPII activity analysis and the other, for TRH content. In this way, we assured reproducibility giving a total number of eight animals per group for each biochemical determination (from two separate trials). Results are expressed as percent of each experiment’s control. 2.3. Determination of PPII activity 2.3.1. Membrane preparation All procedures were performed at 4 8C as previously described (de Gortari et al., 1995). Briefly, frozen brain regions were sonicated (10% w/v) in 50 mM phosphate buffer pH 7.4 and centrifuged for 15 min at 1000 g. Supernatants were recovered in another tube, pellets diluted in half of the original volume of phosphate buffer and centrifuged again. The second supernatant was pooled with the first and centrifuged at 12,000 g for 15 min; pellets were diluted in 200 ml of phosphate buffer. An aliquot of 10 ml was separated for protein determination and the rest used for enzymatic activity measurement. 2.3.2. Fluorometric assay for PPII activity Assay was performed as described (Vargas et al., 1998). Incubation mixture (50 ml) included 6 mg/ml membrane
2.4. Protein determination Ten microliters of the membrane preparation were digested with 1N NaOH for 24 h at room temperature and protein quantified (Lowry et al., 1951). 2.5. PPII and TRH mRNA semi-quantification
2.6. TRH radioimmunoassay (RIA) Tissues homogenized in 500 ml of 20% acetic acid were centrifuged for 15 min at 12,000 g at 4 8C. The supernatant was extracted with methanol (65% final concentration), evaporated and analyzed as described (de Gortari et al., 1995). All reagents were from Sigma, St. Louis, MO, USA. 2.7. Statistical analysis Data of PPII activity and mRNA levels of animal groups sacrificed at different times were compared by a Student’s t-test against its own control sacrificed simultaneously.
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Results from protocol 3 (TRH content and expression, PPII activity and mRNA levels) were analyzed by a two-way ANOVA using as factor 1 the ethanol group and factor 2 drug treatments. This was followed by a Student–Newman–Keuls method post-hoc analysis; significance was considered at p < 0.05.
versus 100 12%), as well as in hippocampus after 2 h (341 70% versus 100 3%) and 16 h (165 19% versus 100 16%). No significant changes were found in amygdala at any time, neither in the above mentioned regions at other times studied (not shown). 3.2. Effect of NMDA-R and GABAA-R agonists and antagonists injection on TRH metabolism
3. Results 3.1. Effects of ethanol on PPII mRNA levels and activities Ethanol administration affected transiently PPII activity in all the regions and with different kinetics. The earliest changes (1 h) were found in frontal cortex (64 7% compared to controls: 100 12%), while other regions varied until 6 h: hippocampus (51 9% versus 100 17%), n. accumbens (62 3% versus 100 13%) and amygdala (73 6% versus 100 8%). Increased activity was found only in hypothalamus after 24 h (153 9.7%, controls: 100 8.8%) (Fig. 1). The levels of PPII mRNA were quantified at various times after ethanol administration, introducing earlier times than those where activity was affected. The only significant changes in PPII mRNA levels were found in frontal cortex at 0.5 h (38 3.4% versus 100 25% in controls) and increasing at 4 h (218 63% versus 100 8% in controls). Decrease in PPII mRNA levels were detected also in n. accumbens at 1 h (63 2% versus 100 16% controls) while in hypothalamus, levels were increased after 4 h (205 27% versus 100 30%) and 16 h (402 67%
To analyze if NMDA or GABAA receptors, could be involved in the changes found on PPII activity or expression, animals were sacrificed 1 h after the administration of ethanol or other drugs to evaluate the most immediate changes trying to avoid indirect effects due to metabolic alterations. This time was chosen because the most striking changes found in PPII activity or its mRNA were observed 1 h after ethanol injection in frontal cortex and n. accumbens; or, in TRH and its mRNA also in hippocampus (de Gortari et al., 2000a). In frontal cortex, PPII activity was decreased by ethanol administration or by MK-801 followed by ethanol; NMDA injection also inhibited PPII activity but when combined with ethanol, inhibition was less pronounced (Table 1). Muscimol or picrotoxin injected alone, had no significant effect on PPII activity but prevented ethanol’s inhibitory effect. The levels of PPII mRNA were decreased by ethanol, MK-801 or MK801 + ethanol and by muscimol. The inhibition produced by ethanol was impeded by NMDA, muscimol or picrotoxin. ProTRH mRNA was not detected in frontal cortex. Although TRH levels (measured by RIA) were significantly increased by MK-801 and
Fig. 1. Kinetics of ethanol effect on PPII activity in brain regions of Wistar rats. Rats were injected i.p. with 3 g/kg of ethanol and sacrificed at 1, 6 and 24 h later. PPII activity was quantified by a fluorometric assay using TRH-bNA as substrate. Results are the mean S.E.M. in percent of control animals (saline injection) sacrificed at the same time (n = 8). Representative control values: amygdala, 4.4 0.6; frontal cortex, 6.6 1; hippocampus, 6.46 1.1; hypothalamus, 2 0.4; n. accumbens, 3 0.42 pmol bNA/min/mg protein. A Student’s t-test was performed to compare each experimental group against its paired control at every time of sacrifice. (*): Significant difference (p < 0.05).
P. de Gortari et al. / Neurochemistry International 46 (2005) 347–356 Table 1 Changes in TRH metabolism in frontal cortex
Control Ethanol NMDA NMDA + ethanol MK-801 MK-801 + ethanol Muscimol Muscimol + ethanol Picrotoxin Picrotoxin + ethanol
TRH (pg (%))
PPII activity (%)
PPII mRNA (%)
100 4.5 82 27 113 40 89 34 158 21 176 30 150 39 135 64 112 11 135 31
100 4 70 13*hi 72 10*hi 83 4hi 88 10hi 79 8*hi 86 7hi 112 5abcde 81 10hi 118 6abcde
100 11 75 1*d 82 8d 91 4c 66 14*f 70 8*d 44 12*abefghi 85 11d 76 11d 81 4d
Male Wistar rats were injected i.p. with ethanol, NMDA, MK-801, muscimol or picrotoxin and sacrificed 60 min later. When drugs were combined with ethanol they were injected 30 min before ethanol injection. TRH contents measured by RIA or PPII activity were quantified in half hemisphere and PPII mRNA levels, in the other half hemisphere. For each parameter, results are the mean of two independent experiments S.E.M. (n = 8). Data are expressed as % of controls (0.9% saline injection) of: PPII activity (pmol bNA/min/mg of protein); PPII mRNA (ratio of PPII amplicon over ciclophilin amplicon arbitrary units); TRH content (pg/tissue). Control values for TRH content are 425 19 pg; for PPII activity are 6.8 0.23 pmol bNA/min/mg of protein. A two-way ANOVA followed by post-hoc analysis using Student–Newman–Keuls method, showed significant differences (p < 0.05) (*) vs. control group, (a) vs. ethanol, (b) vs. NMDA, (c) vs. MK-801, (d) vs. muscimol, (e) vs. picrotoxin, (f) vs. NMDA + ethanol, (g) vs. MK-801 + ethanol, (h) vs. muscimol + ethanol, (i) vs. picrotoxin + ethanol groups. Significant interaction between ethanol and drugs groups for PPII activity was F(4,40) = 7.134, p < 0.001; for TRH content was F(4,20) = 0.320, p = 0.861; for PPII mRNA was F(4,50) = 4.035, p = 0.007.
MK-801 + ethanol according to Student’s t-test, significance was loss by two-way ANOVA (Table 1). In n. accumbens, PPII activity was not modified after 1 h of either ethanol or drugs that affect NMDA or GABA receptors (Fig. 2A). The levels of its mRNA were decreased by ethanol, MK-801 or MK-801 with ethanol, and by muscimol ethanol; picrotoxin prevented the effect of ethanol (Fig. 2A). Ethanol reduced TRH content and this effect was reproduced with MK-801 and muscimol injections either alone or with ethanol. NMDA and picrotoxin reversed ethanol inhibition. TRH mRNA levels increased after ethanol injection; a further increase was observed if ethanol was combined with MK-801 and this effect was mimicked by muscimol and contrarrested by picrotoxin or NMDA (Fig. 2B). In hippocampus, PPII activity or its mRNA were not affected by ethanol at 1 h (not shown). The previously reported increase in TRH content by ethanol (de Gortari et al., 2000a) was reproduced; it was not affected if ethanol injection was preceded by MK-801 or muscimol [162 15*, 154 18*, 212 24* versus 100 5% (204 10 pg/tissue in controls), respectively] while MK-801 (133 5%) or muscimol alone (125 5%) had no effect (*p < 0.05). TRH mRNA levels increased after 1 h of ethanol injection as reported (de Gortari et al., 2000a) (160 5%* versus controls: 100 25%); MK-801 mimicked the effect of
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ethanol (416 5%*) when injected alone or before ethanol (154 4.6%*); NMDA (90 13%), NMDA + ethanol (94 10%), muscimol (81 23%), muscimol + ethanol (80 35%), picrotoxin (96 49%) or picrotoxin + ethanol (72 29%) induced no change (*p < 0.05).
4. Discussion Ethanol induces specific changes on TRH content and its synthesis in the CNS (de Gortari et al., 2000a, 2002). One of the major conclusions coming from the study is that the activity of its degrading ectopeptidase PPII is also regulated by an acute injection of ethanol in a region-specific manner. The dose of ethanol used is that utilized in several reports (Zoeller and Rudeen, 1992; Aversano et al., 2002; Le et al., 1992; Broadbent et al., 2003; Toropainen et al., 1997). Although saline injection is used as control, it produces a mild stress (Herman et al., 1992) not equivalent to that produced by an i.p. ethanol injection that increases further corticosterone serum levels (Madeira and Paula-Barbosa, 1999; Rivier, 1999). Therefore, results most be interpreted as a combined effect of increased stress and ethanol. However, in paradigms where corticosterone is increased, such as fasting (de Gortari et al., 2000b), or exposure to the defensive burying paradigm (preliminary results) main changes in the levels of TRH or its mRNA are observed in amygdala, a region where no immediate changes in TRH or PPII activity were detected. 4.1. Effect of ethanol on PPII activity and mRNA levels Pyroglutamyl aminopeptidase II is an aminopeptidase with exquisite substrate requirements. It degrades peptides with a pglu-X-proNH2 sequence, X being an uncharged, moderately bulky residue (Kelly et al., 2000) and TRH is the only known endogenous substrate. PPII activity was inhibited in membranes prepared from frontal cortex of animals sacrificed 1 h after ethanol administration while in other regions as n. accumbens, amygdala and hippocampus, only after 6 h. This difference in the kinetics of response argues against a general effect such as that of ethanol binding to hydrophobic pockets in PPII causing conformational changes leading to the fast inhibition observed in cortex (Shahidullah et al., 2003); instead, it points for PPII activity regulation mediated through membrane receptors or intracellular cascades. A more definitive argument is that most PPII activity changes (except in hippocampus) were preceded by PPII mRNA levels changes in the same direction. However at 1 h, decreased activity could hardly be explain by regulation of its synthesis but be due either to internalization, as has been reported for another ectoenzyme, NEP (Erdos et al., 1989) or alternatively, to phosphorylation (Suen and Wilk, 1990) as observed for transporters and receptors (Reith et al., 1997; Ferrani-Kile et al., 2003). We have previously reported a fast response of
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Fig. 2. Changes in TRH metabolism in the n. accumbens. Male Wistar rats were injected i.p. with ethanol, NMDA, MK-801, muscimol or picrotoxin and sacrificed 60 min later. When drugs were combined with ethanol they were injected 30 min before ethanol injection. TRH content measured by RIA (B, open bars) or PPII activity by a fluorometric assay (A, open bars) were quantified in half hemisphere and proTRH (B, closed bars) or PPII mRNA levels (A, closed bars), in the other half. Results are the mean of two independent experiments S.E.M. (n = 8). Data are expressed as% of controls (saline injection). Control values: PPII activity, 3.11 0.22 pmol bNA/min/mg protein; PPII mRNA levels, 0.42 0.046 arbitrary units; TRH content, 1110 190 pg; TRH mRNA levels, 0.22 0.02 arbitrary units (ratio of PPII or proTRH amplicon over ciclophilin amplicon mRNA). A two-way ANOVA followed by post-hoc analysis using Student–Newman–Keuls method, showed significant differences (p < 0.05), (*) vs. control group, (a) vs. ethanol, (b) vs. NMDA, (c) vs. MK-801, (d) vs. muscimol, (e) vs. picrotoxin, (f) vs. NMDA + ethanol, (g) vs. MK-801 + ethanol, (h) vs. muscimol + ethanol, (i) vs. picrotoxin + ethanol groups. Significant interaction between ethanol and drugs groups for PPII activity was F(4,20) = 1.690, p = 0.192; for TRH content was F(4,50) = 3.335, p = 0.017; for PPII mRNA was F(4,30) = 11.101, p < 0.001; for TRH mRNA was F(4,60) = 7.928, p < 0.001.
TRHergic neurons to ethanol injection in hippocampus, n. accumbens and frontal cortex deduced by altered levels of TRH or its mRNA (de Gortari et al., 2000a). The response however differed, depending on the region. In n. accumbens, peptide content was decreased while mRNA levels increased, supporting neuronal activation leading to increased synthesis and release (Joseph-Bravo et al., 1998). The decrease in TRH content observed at short times (1 h) is interpreted as increased release since soluble intracellular peptidases do not alter endogenous TRH and
PPII acts only in the extracellular space (Charli et al., 1998). If ectopeptidase’s endocytosis occurs in response to peptide release as for receptors, this mechanism would then be unlikely since PPII activity is not modified in n. accumbens where TRH release is inferred; similarly, ligand induced endocytosis could not explain the fast inhibition of PPII in frontal cortex where no apparent release is observed (peptide levels were not modified after 1 h of ethanol injection). It remains to be studied if PPII is phosphorylated by ethanol (Suen and Wilk, 1990); several protein kinases (PKC, PKA,
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MAPK, fynK) whose regional distribution differ (FerraniKile et al., 2003; Proctor et al., 2003; He et al., 2002; Yaka et al., 2003) are affected by ethanol. An inhibitory effect of ethanol has been reported on pyroglutamyl aminopeptidase activity of synaptosomes prepared from cortex tissue (Mayas et al., 2000) but it is not conclusive how much is due to soluble PPI since the substrate used for detection, pglu-b-naphtylamide, is poorly recognized by PPII (Torres et al., 1986). Transient regulation of PPII mRNA levels was observed in frontal cortex and n. accumbens; levels were decreased 0.5–1 h after ethanol administration; the decreased PPII activity detected at 6 h in n. accumbens could be consequence of previously diminished mRNA levels. Regulation of PPII mRNA levels by TRH has been observed in adenohypophysis and this effect can be reproduced by drugs that affect PKC and PKA activities (Vargas et al., 1994). It is tempting to speculate that the inhibited expression of PPII in n. accumbens could be due to the increased TRH release induced by ethanol after 1 h, or at least, coincident with TRH changes. Activity is later recovered and PPII mRNA levels are higher than controls at 4 and 16 h in frontal cortex and hypothalamus, and at 2 h in hippocampus. This later increase in mRNA levels could add to the list of ethanol altered gene expression due to ethanol’s increase in P-CREB (maximum at 3 h in vitro) after causing translocation to the nucleus of the catalytic PKA subunit, ca, and thereby modifying transcription of various genes (Constantinescu et al., 1999; He et al., 2002). The increased levels of PPII mRNA are reflected in higher activity in hypothalamus but not in hippocampus. A difference in response to ethanol between hippocampus and other regions in for example, c-fos and other IEGs expression (Bachtell et al., 1999; Crankshaw et al., 2003), NMDA receptor phosphorylation or subunit composition, kinases distribution etc. has been reported (Ryabinin et al., 1997; Demarest et al., 1999; Kalluri and Ticku, 2002; Yaka et al., 2003; Hodge and Cox, 1998; Woodward, 1999). 4.2. Effect of NMDA-R and GABAA-R agonists and antagonists injection on TRH metabolism Behavioral changes induced by an ethanol injection are related to activation of GABAAR as well as to the inactivation of NMDAR function (Crews et al., 1996) and GABAR antagonists, as picrotoxin, reduce the narcotic action of ethanol (Khanna et al., 2002; Besheer et al., 2003). The fast response observed in frontal cortex and n. accumbens, important areas in ethanol effects, led us to study the effect of NMDA and GABAA receptors in PPII modulation. Up to now, PPII has been proposed to be in the postsynaptic neuron (Joseph-Bravo et al., 1994; Heuer et al., 2000) and, TRH can alter glutamate and GABA neurotransmission in hippocampus and in cortex (Stocca and Nistri, 1995; Barbieri and Nistri, 1997). Enzyme expression could therefore be affected by ethanol directly through its
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actions on NMDA and GABA receptors present at PPII neurons or, through modifying TRH release that could modulate PPII in the postsynaptic neuron. The only structure where the effects of ethanol on TRH content and mRNA levels (increased synthesis and release) were mimicked by activation of GABA or inhibition of NMDA receptors (and confirmed by picrotoxin contrarresting ethanol effects) was n. accumbens suggesting direct activation of TRHergic neurons by these neurotransmitters (Besheer et al., 2003), as suggested for ethanol action through these two receptors (Wirkner et al., 1999). TRH and its receptor, as well as PPII mRNA and activity, have been described in n. accumbens (Heuer et al., 2000) but no information exists regarding the afferents impinging on TRHergic or PPII neurons. The similar modulation by these neurotransmitters on PPII mRNA levels could be due to their direct effect or, indirect via TRH or dopamine (DA) release (Budygin et al., 2001). Drugs affecting dopaminergic transmission modulate other ectopeptidases as NEP and APN’s activity and mRNA levels (Waters et al., 1997). Ethanol effects on n. accumbens are related not only to motor activity but also in reward mechanisms where dopamine and opioids play important roles. TRH stimulatory role on locomotion may be DA-mediated since TRH increases DA release (although there are controversial reports) (Yamamura et al., 1991; Me´ ndez et al., 1993). TRH could be an additional neurotransmitter involved in reward circuit since cocaine administration can affect peptide content in several brain areas (Jaworska-Feil et al., 1997; Pekary et al., 2002) and TRH injections diminish ethanol consumption but dopamine-D2 receptor antagonists contrarrest this effect (Mason et al., 1997). Cortical neurons are very susceptible to alcohol affecting higher functions as memory and arousal (Aversano et al., 2002). Regulation of PPII in frontal cortex appeared complex since a slight but significant inhibition on PPII activity occurred not only by ethanol but also by NMDA injection while the decreased PPII mRNA levels by ethanol was mimicked by NMDA antagonists or GABA agonists (contrarrested by NMDA and picrotoxin). Alcohol not only affects NMDA or GABAA receptors but ion channels, other receptors, and several neurotransmitters (Faingold et al., 1998; Walter and Messing, 1999; Woodward, 1999). Therefore, other afferents might be activated in this region causing opposite effects for example, NMDA increasing intracellular Ca2+ (that in adenohypophysis inhibits PPII activity and gene expression (Vargas et al., 1998) while the inhibitory effect of alcohol be mediated by other pathways. The lack of effect by NMDA in n. accumbens or hippocampus could be explained by differences in receptor concentration leading to differences in calcium reaching the required inhibitory concentrations. As previously mentioned, i.p. injections cause additional stress; although, ethanol still caused the expected behavioral effects since animals were narcotized. The lack of consistency between ethanol and NMDA or GABA modulation observed in
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frontal cortex in contrast to n. accumbens could reflect combined influences modifying the response. The immediate effects of ethanol or the drugs tested on PPII activity or mRNA are of inhibition, in contrast to the stimulatory effect caused by increased synaptic transmission (early kindling). In kindling, the most responsive regions coincided with their epileptogenic susceptibility (amygdala, hippocampus, frontal cortex) (de Gortari et al., 1995) and ethanol effects were observed mainly in n. accumbens and frontal cortex (this study). Regulation of PPII activity and expression, specific to brain regions where TRH content or mRNA levels are affected, support the concept that PPII might play an important role in the regulation of TRH function. The physiological consequence of PPII inhibited activity could lead to a more efficient TRHergic transmission. The regional specificity in regulation of some events involved in TRH metabolism, depending on the paradigm, gives further support for the relevant role of TRH in behavior.
Acknowledgements We thank the technical support of MVZ Mario Aguilar and Sergio Gonza´ lez Trujillo. This work was partially supported by CONACYT (MRI-35806N and DGAPA IN222603).
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Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions P. de Gortaria,*, F. Romerob, M. Cisnerosb, P. Joseph-Bravob a
Department Neurociencias, Instituto Nacional de Psiquiatrı´a, Ramo´n de la Fuente Mun˜iz, Calzada Me´xico-Xochimilco 101, Col. San Lorenzo Huipulco, C.P. 14370, Me´xico b Department Gene´tica del Desarrollo y Fisiologı´a Molecular, Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, Me´xico Received in revised form 31 March 2004; accepted 12 April 2004 Available online 31 December 2004
Abstract Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABAA receptors’ agonists and antagonists were tested at 1 h (ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol’s inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol’s inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons. # 2004 Elsevier Ltd. All rights reserved. Keywords: Pro-TRH mRNA; PPII regulation; NMDA; GABA
1. Introduction In the central nervous system (CNS), efficient inactivating mechanisms limit the time the neurotransmitters remain in the extracellular fluid. Most classical neurotransmitters are rapidly removed from the synaptic cleft by specific transporters (uptake) (Iversen, 1971). In the case of neuropeptides, the principal mode of inactivation, once released, is their degradation by ectopeptidases that usually have a broad * Corresponding author. Tel.: +52 56 55 28 11x114; fax: +52 56 55 99 80. E-mail address: [email protected] (P. de Gortari). 0197-0186/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2004.11.002
specificity. In general, various enzymes can degrade a given peptide; for example, opioids, cholecystokinin, somatostatin and substance P are degraded by neprilysin (NEP, EC 3.4.24.11; ‘‘enkephalinase’’), aminopeptidase N (APN, E.C. 3.4.11.2) or angiotensin converting enzyme (ACE, E.C. 3.4.15.1) (Csuhai et al., 1995; O’Cuinn et al., 1995; Turner et al., 2001). An exception is pyroglutamyl aminopeptidase II (PPII, EC 3.4.19.6) whose only known endogenous substrate is thyrotropin-releasing hormone (TRH) (Csuhai et al., 1995; O’Cuinn et al., 1995). PPII is enriched in nerve endings and has an heterogeneous localization; PPII mRNA distribution coincides with either one of TRH receptor mRNAs (Charli et al., 1998; Heuer et al., 2000). Evidence accumulates
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supporting the inactivating mechanisms as an additional point of homeostatic control; transporters and ectopeptidases can be regulated at different levels and this regulation can in turn play an active role in neurotransmission (Danbolt, 2001; Horschitz et al., 2001; Reith et al., 1997; Antczak et al., 2001; Charli et al., 1998; Konkoy and Davis, 1996; Heuer et al., 1998). In CNS, the expression and activity of NEP and APN are modulated by acute or chronic administration of neuroleptic drugs that affect dopaminergic transmission, or by chronic alcohol consumption; this regulation differentially modifies the rate of degradation of substance P, met-enkephalin or neurotensin (Konkoy and Davis, 1996; Waters et al., 1995, 1997). Activity of these enzymes differs between alcohol preferring and alcohol avoiding mice and rat lines; administration of NEP and ACE inhibitors decreases alcohol-drinking behavior in mice (Winkler et al., 1998) or rats (Grupp and Chow, 1992). Our group has provided evidence for a stringent control of PPII in the adenohypophysis, a target of TRH in its neuroendocrine role. In cultured adenohypophyseal cells, PPII expression and activity are regulated by its substrate TRH, or by drugs that alter protein kinase C (PKC), protein kinase A (PKA), calcium levels or arachidonic acid cascades (Charli et al., 1998; Baeza et al., 2001; Vargas et al., 1998, 2002a). In vivo, PPII is also modulated by TRH and hormones (Charli et al., 1998; Heuer et al., 1998; Vargas et al., 2002b). Modulation of brain PPII activity was demonstrated in a paradigm that activates TRHergic neurons in limbic areas (amygdaloid kindling) (de Gortari et al., 1995). PPII activity is elevated in hippocampus, amygdala and cortex during kindling development (stage II); while it is decreased in fully kindled rats (stage V), when TRH content and mRNA levels are increased (de Gortari et al., 1995, 1998; Knoblach and Kubek, 1997; Rosen et al., 1994). At this later stage, TRH receptor binding is also diminished (Kubek et al., 1993). TRH is widely distributed in the CNS and its administration causes several behavioral changes that support, together with in vitro experiments, its neuromodulatory role (Horita, 1998; Stocca and Nistri, 1995; Kasparov et al., 1994). TRH has been implicated in arousal (Thompson and Rosen, 2000), locomotor activity (Metcalf and Dettmar, 1981) and autonomic functions (Doong and Yang, 2003). Its administration produces antidepressive, antiepileptic or neuroprotective effects (Horita, 1998; Sattin et al., 1999), and shortens the time of narcosis induced by ethanol and barbiturates (Morzorati and Kubek, 1993; French et al., 1993); improves cognitive function in animals affected by chronic ethanol administration (Khan et al., 1993) and blocks the locomotor suppression induced by high doses of ethanol (Breese et al., 1985). An acute administration of ethanol provokes a fast and transient increase in TRH expression in hippocampus and n. accumbens, and a decrease in frontal cortex. The kinetics of the response is different, not only among regions but also to that observed for proenkephalin mRNA suggesting a specific response (de Gortari et al., 2000a).
The aim of this work was to study the effect of an acute ethanol administration on TRH extracellular inactivation measuring PPII activity and its mRNA levels in limbic regions. Since ethanol inhibits NMDA receptors (NMDAR) and activates GABAA receptors (GABAAR) (Crews et al., 1996; Khanna et al., 2002; Besheer et al., 2003) while kindling progression correlates with the increased activation of NMDAR and with decreased GABA receptors (GABAR) activity (Faingold et al., 1998; Sutula et al., 1996) we measured PPII expression and activity, as well as TRH gene expression, in rats receiving agonists or antagonists of these receptors and their possible modulation of ethanol effects.
2. Experimental procedures 2.1. Animal handling and drug administration Male Wistar rats (400–500 g) from the Institute’s colony were fed (Purina Chow) ad libitum and kept in 07:19-h light:dark cycle and controlled temperature (25 8C). The Ethics Committee and Project Commission of the Instituto Nacional de Psiquiatrı´a approved the experiments and care was taken to avoid unnecessary stress. In order to avoid changes due to circadian rhythms, injections were performed at different times in such a way that animals were sacrificed between 11–12:00 h by decapitation. 2.1.1. Protocol 1: kinetics of ethanol effect on PPII activity Rats were injected i.p. with ethanol (3 g/kg of body weight (b.w.) diluted to 25% in 0.9% saline; Baker, Me´ xico); controls were injected simultaneously with equivalent volumes of saline solution. Injections were performed at 1, 6 or 24 h before the time of sacrifice (11:00 h). Six groups of four rats were formed, one control and one experimental for each time (24 animals); two independent experiments were performed (48 animals, n = 8). PPII activity was measured in cellular membranes extracted from hypothalamus, amygdala, n. accumbens, frontal cortex, and hippocampus as described below. 2.1.2. Protocol 2: kinetics of ethanol effect on PPII mRNA levels Based on the effects on PPII activity, and with the aim to analyze if some of those changes could be due to alterations in expression levels of the PPII gene, we varied the time of ethanol injection previous to sacrifice (0.5 h for frontal cortex and 1, 2, 4 and 16 h for the other regions). Each time had its control group so that 10 groups of 4 rats were formed (2 independent experiments). Total RNA of each region (same as in protocol 1) was extracted and analyzed for PPII mRNA levels by RT-PCR as described below. 2.1.3. Protocol 3: effects of drugs altering glutamate or GABA transmission N-methyl-D-aspartate (NMDA) and its antagonist MK801, muscimol (GABAA agonist) and its antagonist
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picrotoxin, were injected i.p. either alone or 30 min before an ethanol injection. Animals injected with ethanol, muscimol or MK-801 were narcotized almost immediately and started to wake up around the time of sacrifice. Animals were sacrificed 1 h after the ethanol injection or after the single injection of the other drugs. Drugs were used at concentrations recommended in the literature (Aversano et al., 2002; Le et al., 1992; Toropainen et al., 1997; Zarrindast and Mousa-Ahmadi, 1999): N-methyl-D-aspartate (30 mg/kg b.w.); MK-801 (dizocilpine; 0.1 mg/kg b.w.); muscimol, (4 mg/kg b.w.); picrotoxin (5 mg/kg b.w.), each diluted in the same volume (5 ml) of 0.9% saline solution, (purchased from SigmaAldrich, St. Louis, MO, USA). Three experiments were performed, each with four animals per group and a control group injected with saline solution. Frontal cortex, hippocampus and n. accumbens were the brain regions dissected.
protein, 1 mM of protease inhibitors N-ethylmaleimide and bacitracin, 17 mg/ml dipeptidyl aminopeptidase (DAP IV; Sigma-Aldrich, St. Louis, MO, USA). After 5 min preincubation at 37 8C, 10 ml (400 mM final) of TRH-bnaphtylamide (TRH-b-NA) were added as substrate and tubes were further incubated for 15, 30 and 60 min at 37 8C. Reaction was stopped with 250 ml of methanol. b-NA production was detected fluorometrically.
2.2. Tissue collection and sample preparation
Frozen brain regions were homogenized in 4 M guanidine thyocianate (ICN, Aurora, OH, USA) and total RNA extracted as described (Chomczynski and Sacchi, 1987). PPII and TRH mRNA were semi-quantified by reverse transcriptase polymerase chain reaction (RT-PCR) using two different control transcripts: glyceraldehyde 3phosphate dehydrogenase (G3PDH) or cyclophilin. Analysis of proTRH or PPII mRNA levels using G3PDH mRNA as control was performed as reported (de Gortari et al., 2000a; Vargas et al., 1998), except that 15 pmol per tube of G3PDH primers and 25 cycles were used. A 257 bp fragment corresponding to nucleotides 165–422 of the cyclophilin cDNA was also amplified for 23 cycles using 50 pmol per tube of primers whose sequences were: 50 -ACA-TGC-TTGCCA-TCC-AGC-C-30 , antisense: 50 -GGG-GAG-AAAGGA-TTT-GGC-TA-30 . Ten microliters of proTRH or PPII PCR product and 5 ml of either G3PDH or cyclophilin PCR product were separated by a 2% agarose gel (Ultra-pure BioRad, Hercules, CA, USA) electrophoresis; gels were stained with ethidium bromide (1 mg/L) and measured by densitometry (Bio-Rad Fluor-S Multi-Imager System and Multianalyst program). The relative amounts of PPII or TRH mRNA were calculated as the ratio of TRH amplicon over that of control amplicon.
Brains were excised (taking care of cutting the optic nerve before removal from skull, to preserve intact the median eminence), and kept at 70 8C until dissection. Hypothalamus, amygdala, n. accumbens, frontal cortex and hippocampus were hand dissected from frozen brains following Palkovits and Brownstein (1988) coordinates. For protocols 1 and 2, the left and right regions were pooled. For protocol 3, left or right regions were stored separately. This later protocol was performed three times and analyzed as follows: (a) experiment 1: one half of each region was used to measure PPII activity and the other half, for RNA extraction to measure mRNA levels of PPII and TRH; (b) experiment 2: TRH content in one half, and RNA extraction in the other half; (c) experiment 3: one half for PPII activity analysis and the other, for TRH content. In this way, we assured reproducibility giving a total number of eight animals per group for each biochemical determination (from two separate trials). Results are expressed as percent of each experiment’s control. 2.3. Determination of PPII activity 2.3.1. Membrane preparation All procedures were performed at 4 8C as previously described (de Gortari et al., 1995). Briefly, frozen brain regions were sonicated (10% w/v) in 50 mM phosphate buffer pH 7.4 and centrifuged for 15 min at 1000 g. Supernatants were recovered in another tube, pellets diluted in half of the original volume of phosphate buffer and centrifuged again. The second supernatant was pooled with the first and centrifuged at 12,000 g for 15 min; pellets were diluted in 200 ml of phosphate buffer. An aliquot of 10 ml was separated for protein determination and the rest used for enzymatic activity measurement. 2.3.2. Fluorometric assay for PPII activity Assay was performed as described (Vargas et al., 1998). Incubation mixture (50 ml) included 6 mg/ml membrane
2.4. Protein determination Ten microliters of the membrane preparation were digested with 1N NaOH for 24 h at room temperature and protein quantified (Lowry et al., 1951). 2.5. PPII and TRH mRNA semi-quantification
2.6. TRH radioimmunoassay (RIA) Tissues homogenized in 500 ml of 20% acetic acid were centrifuged for 15 min at 12,000 g at 4 8C. The supernatant was extracted with methanol (65% final concentration), evaporated and analyzed as described (de Gortari et al., 1995). All reagents were from Sigma, St. Louis, MO, USA. 2.7. Statistical analysis Data of PPII activity and mRNA levels of animal groups sacrificed at different times were compared by a Student’s t-test against its own control sacrificed simultaneously.
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Results from protocol 3 (TRH content and expression, PPII activity and mRNA levels) were analyzed by a two-way ANOVA using as factor 1 the ethanol group and factor 2 drug treatments. This was followed by a Student–Newman–Keuls method post-hoc analysis; significance was considered at p < 0.05.
versus 100 12%), as well as in hippocampus after 2 h (341 70% versus 100 3%) and 16 h (165 19% versus 100 16%). No significant changes were found in amygdala at any time, neither in the above mentioned regions at other times studied (not shown). 3.2. Effect of NMDA-R and GABAA-R agonists and antagonists injection on TRH metabolism
3. Results 3.1. Effects of ethanol on PPII mRNA levels and activities Ethanol administration affected transiently PPII activity in all the regions and with different kinetics. The earliest changes (1 h) were found in frontal cortex (64 7% compared to controls: 100 12%), while other regions varied until 6 h: hippocampus (51 9% versus 100 17%), n. accumbens (62 3% versus 100 13%) and amygdala (73 6% versus 100 8%). Increased activity was found only in hypothalamus after 24 h (153 9.7%, controls: 100 8.8%) (Fig. 1). The levels of PPII mRNA were quantified at various times after ethanol administration, introducing earlier times than those where activity was affected. The only significant changes in PPII mRNA levels were found in frontal cortex at 0.5 h (38 3.4% versus 100 25% in controls) and increasing at 4 h (218 63% versus 100 8% in controls). Decrease in PPII mRNA levels were detected also in n. accumbens at 1 h (63 2% versus 100 16% controls) while in hypothalamus, levels were increased after 4 h (205 27% versus 100 30%) and 16 h (402 67%
To analyze if NMDA or GABAA receptors, could be involved in the changes found on PPII activity or expression, animals were sacrificed 1 h after the administration of ethanol or other drugs to evaluate the most immediate changes trying to avoid indirect effects due to metabolic alterations. This time was chosen because the most striking changes found in PPII activity or its mRNA were observed 1 h after ethanol injection in frontal cortex and n. accumbens; or, in TRH and its mRNA also in hippocampus (de Gortari et al., 2000a). In frontal cortex, PPII activity was decreased by ethanol administration or by MK-801 followed by ethanol; NMDA injection also inhibited PPII activity but when combined with ethanol, inhibition was less pronounced (Table 1). Muscimol or picrotoxin injected alone, had no significant effect on PPII activity but prevented ethanol’s inhibitory effect. The levels of PPII mRNA were decreased by ethanol, MK-801 or MK801 + ethanol and by muscimol. The inhibition produced by ethanol was impeded by NMDA, muscimol or picrotoxin. ProTRH mRNA was not detected in frontal cortex. Although TRH levels (measured by RIA) were significantly increased by MK-801 and
Fig. 1. Kinetics of ethanol effect on PPII activity in brain regions of Wistar rats. Rats were injected i.p. with 3 g/kg of ethanol and sacrificed at 1, 6 and 24 h later. PPII activity was quantified by a fluorometric assay using TRH-bNA as substrate. Results are the mean S.E.M. in percent of control animals (saline injection) sacrificed at the same time (n = 8). Representative control values: amygdala, 4.4 0.6; frontal cortex, 6.6 1; hippocampus, 6.46 1.1; hypothalamus, 2 0.4; n. accumbens, 3 0.42 pmol bNA/min/mg protein. A Student’s t-test was performed to compare each experimental group against its paired control at every time of sacrifice. (*): Significant difference (p < 0.05).
P. de Gortari et al. / Neurochemistry International 46 (2005) 347–356 Table 1 Changes in TRH metabolism in frontal cortex
Control Ethanol NMDA NMDA + ethanol MK-801 MK-801 + ethanol Muscimol Muscimol + ethanol Picrotoxin Picrotoxin + ethanol
TRH (pg (%))
PPII activity (%)
PPII mRNA (%)
100 4.5 82 27 113 40 89 34 158 21 176 30 150 39 135 64 112 11 135 31
100 4 70 13*hi 72 10*hi 83 4hi 88 10hi 79 8*hi 86 7hi 112 5abcde 81 10hi 118 6abcde
100 11 75 1*d 82 8d 91 4c 66 14*f 70 8*d 44 12*abefghi 85 11d 76 11d 81 4d
Male Wistar rats were injected i.p. with ethanol, NMDA, MK-801, muscimol or picrotoxin and sacrificed 60 min later. When drugs were combined with ethanol they were injected 30 min before ethanol injection. TRH contents measured by RIA or PPII activity were quantified in half hemisphere and PPII mRNA levels, in the other half hemisphere. For each parameter, results are the mean of two independent experiments S.E.M. (n = 8). Data are expressed as % of controls (0.9% saline injection) of: PPII activity (pmol bNA/min/mg of protein); PPII mRNA (ratio of PPII amplicon over ciclophilin amplicon arbitrary units); TRH content (pg/tissue). Control values for TRH content are 425 19 pg; for PPII activity are 6.8 0.23 pmol bNA/min/mg of protein. A two-way ANOVA followed by post-hoc analysis using Student–Newman–Keuls method, showed significant differences (p < 0.05) (*) vs. control group, (a) vs. ethanol, (b) vs. NMDA, (c) vs. MK-801, (d) vs. muscimol, (e) vs. picrotoxin, (f) vs. NMDA + ethanol, (g) vs. MK-801 + ethanol, (h) vs. muscimol + ethanol, (i) vs. picrotoxin + ethanol groups. Significant interaction between ethanol and drugs groups for PPII activity was F(4,40) = 7.134, p < 0.001; for TRH content was F(4,20) = 0.320, p = 0.861; for PPII mRNA was F(4,50) = 4.035, p = 0.007.
MK-801 + ethanol according to Student’s t-test, significance was loss by two-way ANOVA (Table 1). In n. accumbens, PPII activity was not modified after 1 h of either ethanol or drugs that affect NMDA or GABA receptors (Fig. 2A). The levels of its mRNA were decreased by ethanol, MK-801 or MK-801 with ethanol, and by muscimol ethanol; picrotoxin prevented the effect of ethanol (Fig. 2A). Ethanol reduced TRH content and this effect was reproduced with MK-801 and muscimol injections either alone or with ethanol. NMDA and picrotoxin reversed ethanol inhibition. TRH mRNA levels increased after ethanol injection; a further increase was observed if ethanol was combined with MK-801 and this effect was mimicked by muscimol and contrarrested by picrotoxin or NMDA (Fig. 2B). In hippocampus, PPII activity or its mRNA were not affected by ethanol at 1 h (not shown). The previously reported increase in TRH content by ethanol (de Gortari et al., 2000a) was reproduced; it was not affected if ethanol injection was preceded by MK-801 or muscimol [162 15*, 154 18*, 212 24* versus 100 5% (204 10 pg/tissue in controls), respectively] while MK-801 (133 5%) or muscimol alone (125 5%) had no effect (*p < 0.05). TRH mRNA levels increased after 1 h of ethanol injection as reported (de Gortari et al., 2000a) (160 5%* versus controls: 100 25%); MK-801 mimicked the effect of
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ethanol (416 5%*) when injected alone or before ethanol (154 4.6%*); NMDA (90 13%), NMDA + ethanol (94 10%), muscimol (81 23%), muscimol + ethanol (80 35%), picrotoxin (96 49%) or picrotoxin + ethanol (72 29%) induced no change (*p < 0.05).
4. Discussion Ethanol induces specific changes on TRH content and its synthesis in the CNS (de Gortari et al., 2000a, 2002). One of the major conclusions coming from the study is that the activity of its degrading ectopeptidase PPII is also regulated by an acute injection of ethanol in a region-specific manner. The dose of ethanol used is that utilized in several reports (Zoeller and Rudeen, 1992; Aversano et al., 2002; Le et al., 1992; Broadbent et al., 2003; Toropainen et al., 1997). Although saline injection is used as control, it produces a mild stress (Herman et al., 1992) not equivalent to that produced by an i.p. ethanol injection that increases further corticosterone serum levels (Madeira and Paula-Barbosa, 1999; Rivier, 1999). Therefore, results most be interpreted as a combined effect of increased stress and ethanol. However, in paradigms where corticosterone is increased, such as fasting (de Gortari et al., 2000b), or exposure to the defensive burying paradigm (preliminary results) main changes in the levels of TRH or its mRNA are observed in amygdala, a region where no immediate changes in TRH or PPII activity were detected. 4.1. Effect of ethanol on PPII activity and mRNA levels Pyroglutamyl aminopeptidase II is an aminopeptidase with exquisite substrate requirements. It degrades peptides with a pglu-X-proNH2 sequence, X being an uncharged, moderately bulky residue (Kelly et al., 2000) and TRH is the only known endogenous substrate. PPII activity was inhibited in membranes prepared from frontal cortex of animals sacrificed 1 h after ethanol administration while in other regions as n. accumbens, amygdala and hippocampus, only after 6 h. This difference in the kinetics of response argues against a general effect such as that of ethanol binding to hydrophobic pockets in PPII causing conformational changes leading to the fast inhibition observed in cortex (Shahidullah et al., 2003); instead, it points for PPII activity regulation mediated through membrane receptors or intracellular cascades. A more definitive argument is that most PPII activity changes (except in hippocampus) were preceded by PPII mRNA levels changes in the same direction. However at 1 h, decreased activity could hardly be explain by regulation of its synthesis but be due either to internalization, as has been reported for another ectoenzyme, NEP (Erdos et al., 1989) or alternatively, to phosphorylation (Suen and Wilk, 1990) as observed for transporters and receptors (Reith et al., 1997; Ferrani-Kile et al., 2003). We have previously reported a fast response of
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Fig. 2. Changes in TRH metabolism in the n. accumbens. Male Wistar rats were injected i.p. with ethanol, NMDA, MK-801, muscimol or picrotoxin and sacrificed 60 min later. When drugs were combined with ethanol they were injected 30 min before ethanol injection. TRH content measured by RIA (B, open bars) or PPII activity by a fluorometric assay (A, open bars) were quantified in half hemisphere and proTRH (B, closed bars) or PPII mRNA levels (A, closed bars), in the other half. Results are the mean of two independent experiments S.E.M. (n = 8). Data are expressed as% of controls (saline injection). Control values: PPII activity, 3.11 0.22 pmol bNA/min/mg protein; PPII mRNA levels, 0.42 0.046 arbitrary units; TRH content, 1110 190 pg; TRH mRNA levels, 0.22 0.02 arbitrary units (ratio of PPII or proTRH amplicon over ciclophilin amplicon mRNA). A two-way ANOVA followed by post-hoc analysis using Student–Newman–Keuls method, showed significant differences (p < 0.05), (*) vs. control group, (a) vs. ethanol, (b) vs. NMDA, (c) vs. MK-801, (d) vs. muscimol, (e) vs. picrotoxin, (f) vs. NMDA + ethanol, (g) vs. MK-801 + ethanol, (h) vs. muscimol + ethanol, (i) vs. picrotoxin + ethanol groups. Significant interaction between ethanol and drugs groups for PPII activity was F(4,20) = 1.690, p = 0.192; for TRH content was F(4,50) = 3.335, p = 0.017; for PPII mRNA was F(4,30) = 11.101, p < 0.001; for TRH mRNA was F(4,60) = 7.928, p < 0.001.
TRHergic neurons to ethanol injection in hippocampus, n. accumbens and frontal cortex deduced by altered levels of TRH or its mRNA (de Gortari et al., 2000a). The response however differed, depending on the region. In n. accumbens, peptide content was decreased while mRNA levels increased, supporting neuronal activation leading to increased synthesis and release (Joseph-Bravo et al., 1998). The decrease in TRH content observed at short times (1 h) is interpreted as increased release since soluble intracellular peptidases do not alter endogenous TRH and
PPII acts only in the extracellular space (Charli et al., 1998). If ectopeptidase’s endocytosis occurs in response to peptide release as for receptors, this mechanism would then be unlikely since PPII activity is not modified in n. accumbens where TRH release is inferred; similarly, ligand induced endocytosis could not explain the fast inhibition of PPII in frontal cortex where no apparent release is observed (peptide levels were not modified after 1 h of ethanol injection). It remains to be studied if PPII is phosphorylated by ethanol (Suen and Wilk, 1990); several protein kinases (PKC, PKA,
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MAPK, fynK) whose regional distribution differ (FerraniKile et al., 2003; Proctor et al., 2003; He et al., 2002; Yaka et al., 2003) are affected by ethanol. An inhibitory effect of ethanol has been reported on pyroglutamyl aminopeptidase activity of synaptosomes prepared from cortex tissue (Mayas et al., 2000) but it is not conclusive how much is due to soluble PPI since the substrate used for detection, pglu-b-naphtylamide, is poorly recognized by PPII (Torres et al., 1986). Transient regulation of PPII mRNA levels was observed in frontal cortex and n. accumbens; levels were decreased 0.5–1 h after ethanol administration; the decreased PPII activity detected at 6 h in n. accumbens could be consequence of previously diminished mRNA levels. Regulation of PPII mRNA levels by TRH has been observed in adenohypophysis and this effect can be reproduced by drugs that affect PKC and PKA activities (Vargas et al., 1994). It is tempting to speculate that the inhibited expression of PPII in n. accumbens could be due to the increased TRH release induced by ethanol after 1 h, or at least, coincident with TRH changes. Activity is later recovered and PPII mRNA levels are higher than controls at 4 and 16 h in frontal cortex and hypothalamus, and at 2 h in hippocampus. This later increase in mRNA levels could add to the list of ethanol altered gene expression due to ethanol’s increase in P-CREB (maximum at 3 h in vitro) after causing translocation to the nucleus of the catalytic PKA subunit, ca, and thereby modifying transcription of various genes (Constantinescu et al., 1999; He et al., 2002). The increased levels of PPII mRNA are reflected in higher activity in hypothalamus but not in hippocampus. A difference in response to ethanol between hippocampus and other regions in for example, c-fos and other IEGs expression (Bachtell et al., 1999; Crankshaw et al., 2003), NMDA receptor phosphorylation or subunit composition, kinases distribution etc. has been reported (Ryabinin et al., 1997; Demarest et al., 1999; Kalluri and Ticku, 2002; Yaka et al., 2003; Hodge and Cox, 1998; Woodward, 1999). 4.2. Effect of NMDA-R and GABAA-R agonists and antagonists injection on TRH metabolism Behavioral changes induced by an ethanol injection are related to activation of GABAAR as well as to the inactivation of NMDAR function (Crews et al., 1996) and GABAR antagonists, as picrotoxin, reduce the narcotic action of ethanol (Khanna et al., 2002; Besheer et al., 2003). The fast response observed in frontal cortex and n. accumbens, important areas in ethanol effects, led us to study the effect of NMDA and GABAA receptors in PPII modulation. Up to now, PPII has been proposed to be in the postsynaptic neuron (Joseph-Bravo et al., 1994; Heuer et al., 2000) and, TRH can alter glutamate and GABA neurotransmission in hippocampus and in cortex (Stocca and Nistri, 1995; Barbieri and Nistri, 1997). Enzyme expression could therefore be affected by ethanol directly through its
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actions on NMDA and GABA receptors present at PPII neurons or, through modifying TRH release that could modulate PPII in the postsynaptic neuron. The only structure where the effects of ethanol on TRH content and mRNA levels (increased synthesis and release) were mimicked by activation of GABA or inhibition of NMDA receptors (and confirmed by picrotoxin contrarresting ethanol effects) was n. accumbens suggesting direct activation of TRHergic neurons by these neurotransmitters (Besheer et al., 2003), as suggested for ethanol action through these two receptors (Wirkner et al., 1999). TRH and its receptor, as well as PPII mRNA and activity, have been described in n. accumbens (Heuer et al., 2000) but no information exists regarding the afferents impinging on TRHergic or PPII neurons. The similar modulation by these neurotransmitters on PPII mRNA levels could be due to their direct effect or, indirect via TRH or dopamine (DA) release (Budygin et al., 2001). Drugs affecting dopaminergic transmission modulate other ectopeptidases as NEP and APN’s activity and mRNA levels (Waters et al., 1997). Ethanol effects on n. accumbens are related not only to motor activity but also in reward mechanisms where dopamine and opioids play important roles. TRH stimulatory role on locomotion may be DA-mediated since TRH increases DA release (although there are controversial reports) (Yamamura et al., 1991; Me´ ndez et al., 1993). TRH could be an additional neurotransmitter involved in reward circuit since cocaine administration can affect peptide content in several brain areas (Jaworska-Feil et al., 1997; Pekary et al., 2002) and TRH injections diminish ethanol consumption but dopamine-D2 receptor antagonists contrarrest this effect (Mason et al., 1997). Cortical neurons are very susceptible to alcohol affecting higher functions as memory and arousal (Aversano et al., 2002). Regulation of PPII in frontal cortex appeared complex since a slight but significant inhibition on PPII activity occurred not only by ethanol but also by NMDA injection while the decreased PPII mRNA levels by ethanol was mimicked by NMDA antagonists or GABA agonists (contrarrested by NMDA and picrotoxin). Alcohol not only affects NMDA or GABAA receptors but ion channels, other receptors, and several neurotransmitters (Faingold et al., 1998; Walter and Messing, 1999; Woodward, 1999). Therefore, other afferents might be activated in this region causing opposite effects for example, NMDA increasing intracellular Ca2+ (that in adenohypophysis inhibits PPII activity and gene expression (Vargas et al., 1998) while the inhibitory effect of alcohol be mediated by other pathways. The lack of effect by NMDA in n. accumbens or hippocampus could be explained by differences in receptor concentration leading to differences in calcium reaching the required inhibitory concentrations. As previously mentioned, i.p. injections cause additional stress; although, ethanol still caused the expected behavioral effects since animals were narcotized. The lack of consistency between ethanol and NMDA or GABA modulation observed in
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frontal cortex in contrast to n. accumbens could reflect combined influences modifying the response. The immediate effects of ethanol or the drugs tested on PPII activity or mRNA are of inhibition, in contrast to the stimulatory effect caused by increased synaptic transmission (early kindling). In kindling, the most responsive regions coincided with their epileptogenic susceptibility (amygdala, hippocampus, frontal cortex) (de Gortari et al., 1995) and ethanol effects were observed mainly in n. accumbens and frontal cortex (this study). Regulation of PPII activity and expression, specific to brain regions where TRH content or mRNA levels are affected, support the concept that PPII might play an important role in the regulation of TRH function. The physiological consequence of PPII inhibited activity could lead to a more efficient TRHergic transmission. The regional specificity in regulation of some events involved in TRH metabolism, depending on the paradigm, gives further support for the relevant role of TRH in behavior.
Acknowledgements We thank the technical support of MVZ Mario Aguilar and Sergio Gonza´ lez Trujillo. This work was partially supported by CONACYT (MRI-35806N and DGAPA IN222603).
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