Differential effect of kinase A and C blockers on lordosis facilitation by progesterone and its metabolites in ovariectomized estrogen-primed rats

Hormones and Behavior 49 (2006) 398 – 404 www.elsevier.com/locate/yhbeh

Differential effect of kinase A and C blockers on lordosis facilitation by progesterone and its metabolites in ovariectomized estrogen-primed rats Oscar González-Flores a,⁎, Juan Manuel Ramírez-Orduña b , Francisco Javier Lima-Hernández a , Marcos García-Juárez a , Carlos Beyer a a

Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala, Apartado Postal 64, c.p. 90000 Tlaxcala, Tlaxcala, Mexico b Departamento de Zootecnia, Universidad Autónoma de Baja California Sur, Mexico Received 12 February 2005; revised 27 August 2005; accepted 30 August 2005 Available online 27 October 2005

Abstract Dose response curves for lordosis behavior was obtained for progesterone (P) and its two ring A–reduced metabolites: 5α-pregnanedione (α-DHP) and 5α,3α-pregnanolone (5α,3α-Pgl) by infusing these progestins in the right lateral ventricle (rlv) of ovariectomized (ovx) estradiol–treated rats (2 μg estradiol benzoate; EB), 40 h before intracerebro–ventricular (icv) injection. Effective doses 50 (ED50) revealed that ring A–reduced progestins were more potent than P itself to induce lordosis behavior. Two dose levels, one producing the maximal effect and the other one producing a submaximal response (ED50–ED60), were selected for testing the capacity of RpAMPS, a kinase A blocker, and H7, a kinase C blocker, to modify the response to the three progestins. rlv injection of RpAMPS significantly depressed the lordosis response to the two dose levels of P and α-DHP but failed to significantly inhibit that of 5α,3α-Pgl. The administration of H7 prevented the effect of both 5α–reduced progestins without affecting the response to P. The results suggest that P and its ring A–reduced metabolites stimulate lordosis behavior through different cellular mechanisms: P acting mainly through the cAMP-kinase system; α-DHP through both kinase A and kinase C signaling pathways and 5α,3α-Pgl through the kinase C system. © 2005 Elsevier Inc. All rights reserved. Keywords: Progesterone; Ring A reduced progestins; RpAMPS; H7; Lordosis behavior; Progesterone receptor; Kinase A; Kinase C; cAMP; MAPK

Introduction In estrogen-primed rodents, the interaction of progesterone (P) with an intracellular receptor (P receptor; PR) is essential for the facilitation of estrous behavior (lordosis and proceptivity; Blaustein and Feder, 1979, 1980; Blaustein, 1982; Etgen, 1984; Moguilewsky and Raynaud, 1979). Yet, ring A-reduced metabolites of P, i.e., 5α-pregnanedione (α-DHP), and 5α,3αpregnanolone (5α,3α-Pgl), with low or null affinity for the PR, are more potent than P itself when given intravenously (i.v.) or intracerebro ventricular (icv) to facilitate lordosis behavior in ovariectomized (ovx) estradiol (E2)-primed rats (Beyer et al., 1989, 1995; Rodríguez-Manzo et al., 1986). This suggests that ring A-reduced progestins act through a different cellular mechanism than P. Surprisingly, the behavioral effect of these progestins requires the participation of the PR, since ⁎ Corresponding author. E-mail address: [email protected] (O. González-Flores). 0018-506X/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2005.08.011

administration of the antiprogestin RU486 prevents their action (Beyer et al., 1995; González-Mariscal et al., 1989). This finding indicates that, directly or indirectly, ring A-reduced P metabolites act on the PR to facilitate lordosis. Ring A-reduced progestins act at the synaptic level to modulate the action of several neurotransmitters such as GABA (Harrison and Simmonds, 1984; Majewska et al., 1986), glycine (Prince and Simmonds, 1992), glutamate (Park-Chung et al., 1994), noradrenaline (Belmar et al., 1998; Kubli-Garfias et al., 1983), opiates (Reddy and Kulkarni, 1997; Winfree et al., 1992) and adenosine (Akhondzadeh and Stone, 1995; Mandhane et al., 1999). Some of these neurotransmitters are most likely present in synapses of the complex neural circuitry involved in the expression of lordosis behavior. Therefore, a plausible explanation for the action of ring A-reduced progestins on lordosis behavior is that they modulate the effect of neurotransmitters on their membrane receptors. Recent results suggest that P or its ring A-reduced metabolites (5α,3α-Pgl) facilitate lordosis in the

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rat (Petralia and Frye, 2004) and the hamster (Sumida et al., 2005) by modulating the activity of dopamine 1 receptors in the ventral tegmental area (VTA). It is also probable that progestins can act directly on the membrane of neurons to trigger second messengers such as cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) (Beyer and González-Mariscal, 1986; Chu et al., 1999; Collado et al., 1985; González-Flores and Etgen, 2004; González-Flores et al., 2004; Mani et al., 2000; Whalen and Lauber, 1986), that could influence the PR or its associated molecules (coactivators, corepressors, etc.). The participation of second messengers in the facilitation of lordosis behavior was suggested by studies showing that cAMP or cGMP administration to E2-primed rats facilitates lordosis behavior (Beyer et al., 1981, 1982; Beyer and GonzálezMariscal, 1986; Chu and Etgen, 1997; Chu et al., 1999; Fernández-Guasti et al., 1983). The possibility that P or its metabolites could act through G protein-associated processes to elicit lordosis behavior was initially shown by Collado et al. (1985) who found, associated to the expression of this behavior, an increase in cAMP levels in the ventromedial hypothalamus (VMH) and medial preoptic area (mPOA) of ovx rats treated with EB and P. Activation of the cAMP-kinase-A system was also found by Mani et al. (2000). These last workers also found that RpAMPS, a kinase blocker, decreased the facilitatory effect of P on the lordosis behavior of EB ovxtreated rats. Similarly, blocking the cAMP-adenylate cyclase pathway prevented the stimulatory effect of the infusion of 5α,3α-Pgl in the VTA on the lordosis behavior of ovx rats (Petralia and Frye, 2004). The role of kinase C in lordosis behavior has also been suggested by the facilitation of lordosis induced by phorbol esters in ovx EB-primed rats (Kow et al., 1994a; Mobbs et al., 1989). Phorbol esters mimic the action of diacylglycerol on kinase C, a signaling system activated by neurotransmitters such as acetylcholine and noradrenaline which are known to facilitate lordosis behavior in ovx EBprimed rats (Clemens et al., 1980; Etgen et al., 1992; Fernández-Guasti et al., 1985; Vathy and Etgen, 1989). In the present study, we tested the effect of blockers of kinase A and kinase C on the facilitatory effect of lordosis behavior by P and its two immediate ring A-reduced metabolites: α-DHP and 5α,3α-Pgl. This information will permit us to establish if P and its ring A-reduced metabolites act through the same or different cellular mechanism. Methods A total of 429 subjects (Ss) were used in this study. Ss were sexually inexperienced female Wistar rats (240–280 g) bred in our colony. They were kept at 23 ± 2°C with an inverted light–dark cycle (14 h light, 10 h dark, lights on at 2300 h). Ss were fed with Purina rat chow and water ad libitum. Ss were bilaterally ovx under ether anesthesia, injected with penicillin (22,000 u.i./kg) and housed in collective cages (4 Ss per cage). Two weeks later, they were anesthetized with xylazine (4 mg/kg) and ketamine (80 mg/kg) and placed in a Kopf stereotaxic instrument (Tujunga, California). Ss were implanted with a stainless steel cannula (22 gauge, 17 mm long) in the right lateral ventricle (rlv), coordinates; interaural 8.20 mm, bregma 0.80 mm (Paxinos and Watson, 1997). A stainless steel screw was fixed to the skull and both cannula and screw were attached to the bone with dental cement. An insert cannula (30 gauge)

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provided with a cap was introduced into the guide cannula to prevent clogging and contamination. Animal care and all the experimental procedures adhered to the Mexican Law for the Protection of Animals.

Experimental procedure Experiment 1 Establishment of dose–response curves and effective doses 50 (ED50) for P, α-DHP and 5α,3α-Pgl administered into the rlv route to EB (2 μg)-treated Ss. One week after surgery, 157 Ss were injected s.c. with EB (2 μg) and 40 h later with the progestins (P, α-DHP or 5α,3α-Pgl). Progestins were infused into rlv through a plastic Clay Adams catheter (PE 10 No 7401), fitted to a Hamilton Syringe (10 μl) inserted into the guide cannula. Steroids were purchased from Sigma (St. Louis MO, USA). Progestins were dissolved in sesame oil (1 μl volume). This vehicle has been previously used for icv infusions of steroids (Beyer et al., 1989; González-Flores and Etgen, 2004; González-Mariscal et al., 1989; Mani et al., 1994). Dosages explored were: P; 1.3 ng (n = 8), 5 ng (n = 8), 9 ng (n = 8), 13 ng (n = 8), 130 ng (n = 8), 1300 ng (n = 8), 13,000 ng (n = 9); α-DHP; 0.32 ng (n = 9), 1.3 ng (n = 7), 5 ng (n = 8), 9 ng (n = 6), 13 ng (n = 7), 130 ng (n = 8) and 5α,3α-Pgl, 1.3 ng (n = 10), 5 ng (n = 11), 9 ng (n = 8), 13 ng (n = 9) and 130 ng (n = 9). Control injections (vehicle, n = 8) were also performed. These time intervals were selected considering that maximal response to the progestins used is found between 2 and 4 h (Beyer et al., 1995). Ss were used only once. Ss infused outside the rlv were excluded from the study. Behavioral testing Females were placed in a circular plexiglas arena (53 cm in diameter) with a vigorous male. Receptivity for each female was determined as a lordosis quotient [LQ = (number of lordosis / 10 mounts) × 100]. The intensity of lordosis (0 to 3) was quantified according to the lordosis score (LS) proposed by Hardy and DeBold (1972). These testing intervals were used since in our rats, maximal response to progestin injection is found between 2 and 4 h. Experiment 2 Effect of RpAMPS on the effectiveness of progestins for eliciting lordosis behavior. RpAMPS (Rp-adenosine 3′,5′-cyclic monophosphorothiate triethylamonium salt) is a specific inhibitor of kinase A (Gjertsen et al., 1995). This drug has been found effective in blocking the cAMP signal by inhibiting protein kinase-A (PKA) “in vivo” (Rothermel et al., 1984). Moreover, Mani et al. (2000) reported that the icv injection of RpAMPS to ovx EB-primed rats interfered with the lordosis response induced by P. Therefore, this drug was selected to asses the role of cAMP and PKA in the facilitation of lordosis by P and its metabolites. A preliminary study using this treatment revealed that it does not produce unspecific effects which could confound interpretation of the results. This drug was purchased from Sigma (St. Louis MO, USA). One week after rlv implantation of cannula, 134 rats were treated with EB (2 μg) and 40 h later with one of the progestins (P, α-DHP, 5α,3α-Pgl). Two dose levels for each progestin were selected from experiment 1. One dose was maximal, i.e., the one producing the maximal effect for that progestin, and a submaximal dose (between 50 and 60 ED). Doses for P were: 9 and 130 ng; for α-DHP, 1.3 and 9 ng; and for 5 α,3α-Pgl, 5 and 13 ng. Five minutes before and 15 min after the injection of the progestins, RpAMPS was injected (100 ng) into the rlv. Behavioral observations were performed at the same time intervals than in experiment 1. Experiment 3 Effect of H7 on lordosis behavior induced by two selected doses of P, α-DHP and 5α,3α-Pgl. H7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) is a potent inhibitor of kinase C and to a lesser extent of kinase A (c-AMP dependent protein kinase; Hidaka et al., 1984). This drug has been used in several “in vivo” studies in the rat (Jaw et al., 1993; Narita et al., 1994; Tokuyama et al., 1995; Valverde et al., 1996). A dose of 3.6 ng was selected from previous studies showing its effectiveness “in vivo” for blocking kinase C

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(Jaw et al., 1993; Narita et al., 1994; Tokuyama et al., 1995; Valverde et al., 1996) and by our finding that two rlv injections of this dose to ovx rats did not produce significant toxic effects, as evidenced by its failure to alter locomotion, grooming behavior and exploratory behavior for a 2 h period. This drug was purchased from Sigma (St. Louis MO, USA). One week after rlv implantation of cannula, 138 rats were injected with 2 μg of EB (h 0) and 40 h later progestins were infused into the ventricle. Doses for progestins were the same as those used in experiment 2. H7 or its vehicle (distilled water, 1 μl) was injected 5 min before and 15 min after the administration of the progestins. Behavioral tests (lordosis) were performed as in experiment 1 at 2 and 4 h post-progestin injection.

Histological study Twenty-four hours after completion of the experiments, Ss were anesthetized with ether and 1% methylene blue was administered through the cannula. The brain was removed and sectioned in the transverse plane to check the cannula position in the rlv. The animals whose cannula was not in the ventricle were discarded from the experiment. Statistical analysis In experiment 1, relative potencies of the progestins to elicit lordosis were calculated according to Tallarida and Murray (1987). This method involved the calculation of regression lines, analysis of parallelism, determination of a common slope and calculation of the potency of each progestin in relation to P (reference drug, with relative potency = 1). Criterion for accepting parallelism of the various curves was established at a 95% confidence interval. The effect of the different kinase blockers on the stimulatory effect on lordosis action of the three progestins (experiments 2 and 3) was assessed by comparing the LQs obtained with the progestins alone with those obtained with the progestins plus H7 or RpAMPS. A Mann–Whitney test was used to compare progestins vs. progestin plus kinase blockers groups (Bruning and Kintz, 1987). Probability rejection level was selected at 0.05.

Results Experiment 1. Establishment of dose–response curves and effective doses 50 (ED50) for P, α-DHP and 5α,3α-Pgl administered into the rlv route to EB (2 μg)-treated Ss Fig. 1 shows the log dose–response curves for lordosis behavior (LQ) obtained by the rlv infusion of P and its two ring A-reduced metabolites (α-DHP, 5α,3α-Pgl) at 4 h. P showed significant differences from the control values at the 9 ng dose, while both α-DHP and 5α,3α-Pgl did it at 1.3 ng. Significant lordosis behavior was already observed at 2 h post-injection (not shown) but maximal responses were recorded at 4 h postinjection. Although the responses were not linear across the wide range of dosages employed (Fig. 1), a linear part of the response occurred in the three progestins, allowing regression analysis and the establishment of ED values. ED 50 values were as follows: P 5 ng, α-DHP 1 ng and 5α,3α-Pgl 1 ng. Analysis of parallelism among the three progestins showed that P and αDHP were parallel to each other (T.C. = 41; df = 65, P < 0.05) but not to 5α,3α-Pgl. α-DHP was 9.6 times more potent than P (reference value = 1). This value was significant (P < 0.05). αDHP and 5α,3α-Pgl values were not significantly different. The progestin showing greater efficacy, i.e., that producing the largest response, was 5α,3α-Pgl, while P and α-DHP had similar efficacies. Dose–response curves for α-DHP and 5α,3α-Pgl were dualistic, i.e., large dosages inducing smaller responses. Experiment 2. Effect of RpAMPS on the stimulatory effect of P, α-DHP, and 5α,3α-Pgl on lordosis behavior of ovx EB-treated rats Fig. 2 shows the effect on lordosis behavior of the rlv administration of two selected doses of P, α-DHP and 5α,3αPgl. Administration of RpAMPS significantly decreased the behavioral response to P and α-DHP at 2 h, both in LQ and LS (see Fig. 2 for probability values). Values obtained with RpAMPS alone were not different from those obtained with the vehicle; i.e., without P. The inhibitory effect of RpAMPS was

Fig. 1. Effect of the rlv injection of several doses (1.3 to 130 ng) of progesterone (P), 5α-pregnanedione (α-DHP) or 5α,3α-pregnanolone (5α,3α-Pgl) to ovx estrogenprimed rats on the lordosis quotient.

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Fig. 2. Effect of the rlv injection of 100 ng of RpAMPS on the stimulatory effect of P (9 and 130 ng), α-DHP (1.3 and 9 ng) and 5α,3α-Pgl (5 and 13 ng) on lordosis behavior of ovx EB-treated rats. The facilitation of lordosis by P and αDHP at 2 h was inhibited by RpAMPS. **P < 0.001; *P < 0.005; +P < 0.01 vs. corresponding group receiving progestins plus vehicle.

transitory since at 4 h after progestins injection no significant differences between RpAMPS-treated groups and those receiving only P or α-DHP were noted (Table 1). RpAMPS induced a slight but not significant reduction in the response to 5α,3α-Pgl (LQ). Experiment 3. Effect of H7 on the stimulatory effect of P, α-DHP and 5α,3α-Pgl on lordosis behavior of ovx EB-treated rats Fig. 3 shows LQ values obtained by the administration of two doses of P, α-DHP and 5α,3α-Pgl in combination with vehicle or H7, a kinase C inhibitor. H7 administration failed to interfere with the stimulatory effect of P on lordosis behavior. On the other hand, H7 significantly inhibited the stimulatory effect of αDHP and 5α,3α-Pgl at 2 h after progestin injection (see Fig. 3 for probability values), though full recovery of the lordotic response was noted 4 h post-progestin injection (Table 1). Discussion The present results show that the rlv infusion of P or its metabolites (ring A-reduced) elicits lordosis behavior in ovx rats pretreated with EB. In agreement with previous data using the i.v. route, ring A-reduced metabolites were more potent than P itself for stimulating lordosis behavior (Beyer et al., 1995).

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Fig. 3. Effect of the rlv injection of 3.6 ng of H7 on the stimulatory effect of P (9 and 130 ng), α-DHP (1.3 and 9 ng) and 5α,3α-Pgl (5 and 13 ng) on lordosis behavior of ovx EB-treated rats. The facilitation of lordosis by α-DHP and 5α,3α-Pgl at 2 h was inhibited by H7. **P < 0.001; *P < 0.005 vs. corresponding group receiving progestins plus vehicle.

Although, the effect of both P and its ring A-reduced metabolites on estrous behavior requires their interaction with the PR (Beyer et al., 1995; González-Mariscal et al., 1989), no correlation exists between their affinity for the receptor and their potency for eliciting lordosis behavior. Thus, P which was the least potent of the progestins used has the highest binding affinity for the PR, while 5α,3α-Pgl (the most potent one) lacks affinity for this molecule (Karavolas et al., 1984; Kontula et al., 1975). This suggests that 5α,3α-Pgl interacts indirectly with the PR, through non-ligand mechanisms (González-Flores and Etgen, 2004; González-Flores et al., 2004). Indeed, parallel line assay showed that the dose–response curve of 5α,3α-Pgl was not parallel with those of P and α-DHP, a finding supporting the abovementioned idea. This idea was further supported by the differential sensitivity of the progestins to the two kinase blockers used in this study. The finding that RpAMPS, a blocker of the cAMP-kinase A cascade, inhibited the facilitatory effect of P on lordosis behavior agrees with the results of Mani et al. (2000). This result is also consistent with previous studies showing the participation of cAMP on the production of estrous behavior in the rat. Thus, administration of cAMP analogs either systemically or in the VMH induces lordosis behavior in ovx estrogen-primed rats (Beyer et al., 1981; Beyer and González-Mariscal, 1986). Moreover, injection of phosphodiesterase blockers preventing the degradation of cAMP such as MIX (Methyl isobutyl xanthine) or theophylline enhances and prolongs the effect of P on estrous behavior (Beyer et al., 1982). Additionally, indirect

Table 1 Effect at 4 h of RpAMPS and H7 on lordosis behavior induced by P, α-DHP or 5α,3α-Pgl in ovx estrogen-primed rats α-DHP

P

Vehicle RpAMPS H7

5α,3α-Pgl

9 ng (LQ ± SE)

130 ng (LQ ± SE)

1.3 ng (LQ ± SE)

9 ng (LQ ± SE)

5 ng (LQ ± SE)

13 ng (LQ ± SE)

62 ± 10 71 ± 7 77 ± 6

56 ± 6 71 ± 9 65 ± 12

44 ± 13 63 ± 6 62 ± 9

68 ± 11 60 ± 7 62 ± 7

61 ± 7 59 ± 12 52 ± 13

76 ± 6 76 ± 9 50 ± 11

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evidence that an increase in cAMP is important for lordosis facilitation by P comes from studies showing that the administration of P to ovx estrogen-treated rats produces an increase in both brain cAMP (Collado et al., 1985; Mani et al., 2000) and in kinase A (Mani et al., 2000). The cAMP-kinase A cascade apparently does not act on the PR, since none of the phosphorylating sites reported in this molecule is phosphorylated by kinase A (Chauchereau et al., 1994). A nongenomic mechanism proposed for the action of the cAMPkinase A cascade on the display of estrous behavior involves the phosphorylation activation of DARPP-32 (Mani et al., 2000), a molecule which inhibits phosphatases, thus increasing phosphorylation of many proteins (Svenningsson et al., 2004). The nature of these proteins is not known though they could be those resulting from the transcriptional effect of the PR-P complex itself. Moreover, it is also possible that the PRassociated coactivators which are essential for the full transcriptional activity of the PR are involved in this response (Edwards et al., 2002; Li et al., 2004; Rowan and O’Malley, 2000). Our present findings show that the cAMP-kinase-A system is also related to the stimulatory effect of α-DHP on estrous behavior since RpAMPS significantly depressed the stimulatory effect of this progestin on this behavior. The possibility that α-DHP acts through the some mechanisms as P is further supported by their parallel dose–response curves. Yet, it is somewhat puzzling that α-DHP was significantly more potent than P itself, considering that it has a much lower affinity for the PR than its parent hormone (Kontula et al., 1975; Smith et al., 1974). This finding suggests that, besides binding to the PR, α-DHP activates additional mechanisms for lordosis facilitation. It appears probable that this activation could occur through its reduction to 5α,3α-Pgl, a process readily occurring in brain tissue (Cheng and Karavolas, 1973; Karavolas et al., 1984). RpAMPS failed to significantly inhibit the effect of 5α,3α-Pgl on lordosis behavior. The possibility that 5α,3α-Pgl acts on lordosis behavior through a different mechanism than P was supported by the fact that RpAMPS failed to inhibit its action and that its dose–response curve did not show parallelism with those of P and α-DHP. It appears likely that 5α,3α-Pgl acts on lordosis behavior by various mechanism and at multiple sites, i.e., the VTA and the VMH. Thus, DeBold and Malsbury (1989) and Frye and DeBold (1993) suggested that 5α,3α-Pgl facilitates lordosis behavior in the hamster by acting in the VTA, a region devoid of PR. They suggested that the effect of this progestin on estrous behavior was mediated through its well-known effect on the GABA-A receptor. Activation of GABA-A synapses in the VMH facilitates lordosis behavior in ovx estrogen-primed rats (McCarthy et al., 1990). More recently, it has been proposed that besides its stimulatory action on the GABA-A receptor 5α,3α-Pgl pregnanolone can modulate D1 receptors in VTA neurons thus partially facilitating lordosis behavior both in rats (Petralia and Frye, 2004) and hamsters (Sumida et al., 2005). The diversity of cellular mechanisms potentially participating in the production of lordosis behavior in the rat was revealed by the administration of H7, a drug blocking mainly

kinase C, though having also some effects on kinase A (Hidaka et al., 1984). In spite of this last action, H7 failed to interfere with the facilitatory effect of P on estrous behavior suggesting that its inhibitory effect on kinase A is not as potent as that of RpAMPS. By contrast, H7 depressed the action of α-DHP at both dose levels. This suggests that the action of α-DHP involves the participation of a kinase C pathway besides the cAMP-kinase A cascade. This dual mode of action of α-DHP is consistent with the observation of its greater potency than P. The drastic inhibition of the effect of 5α,3α-Pgl by H7 combined with the failure of RpAMPS to inhibit it, strongly suggests that 5α,3α-Pgl acts mainly, through cellular mechanisms modulated by the kinase C cascade. The role of this system in the expression of estrous behavior is supported by the fact that activation of kinase C by phorbol ester facilitates lordosis in estrogen-primed rats (Kow et al., 1994a,b). Kinase C can be activated both by Ca++ and/or diacylglycerol rises, conditions induced by a large number of agents including neurotransmitters (Agranoff and Fisher, 1994), some of which (e.g., Acetylcholine, GABA; Clemens et al., 1980, 1989; McCarthy et al., 1990) have been reported to induce estrous behavior in ovx estrogen-primed rodents. Our present results suggests that P and its ring A-reduced metabolites use different signaling mechanisms for facilitating lordosis behavior (Beyer and González-Mariscal, 1986; Etgen, 1984; Kow et al., 1994b; Mani et al., 2000) Thus, P appears to use mainly the cAMP-kinase A cascade while 5α,3α-Pgl acts through the kinase C system. Interestingly, α-DHP apparently acts through both signaling systems. Yet, this does not indicate that P acts exclusively through a single second messenger system to induce lordosis behavior. Recent studies indicate that P also interacts with other signaling mechanisms (cGMP, mitogen-activated protein kinase (MAPK); González-Flores and Etgen, 2004; González-Flores et al., 2004) to facilitate lordosis, since blockage of any of these systems significantly depresses lordosis behavior. P and its ring A-reduced metabolites can stimulate lordosis behavior by acting on several brain sites. Thus, progestins have been found to facilitate lordosis in ovx EB-primed rats when implanted or infused into the VMH (Etgen and Barfield, 1986; González-Mariscal et al., 1989), the mPOA (Beyer et al., 1989; Powers, 1972; Rodríguez-Manzo et al., 1986), the VTA (Frye and DeBold, 1993; Lisciotto and DeBold, 1991) and the mesencephalic tegmentum (Ross et al., 1971). Interestingly, progestins may use different cellular mechanism to facilitate lordosis: (1) directly interacting with the intracellular RP; (2) modulating neurotransmitter release or their effect on receptors and (3) directly activating membrane receptors or altering membrane physicochemical characteristics (Boonyaratanakornkit et al., 2001; Edwards et al., 2002). These actions may be exerted on the same or different neurons, making the analysis of the cellular mechanism underlying lordosis a complicated process. Since in the present study we used rlv infusions, it is not possible to establish the anatomical sites of action of either progestins or kinase blockers. Further studies using restricted local infusions in selected areas must be done to unravel the complex organization of this system.

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