Adenosine A1 and A2a receptors modulate the nitrergic system in cell culture from dorsomedial medulla oblongata

M.A. Costa a, J.P.P. Matsumoto a, D.C. Carrettiero b, D.R. Fior-Chadi a,*

Abstract

Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the interaction between adenosine and NO is well known in the periphery, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, related to neural control of circulation, are not completely understood and might be relevant for individuals predisposed to hypertension. In this study we evaluate the interaction between adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Using quantification of nitrite levels, RT-PCR analysis and RNA interference we demonstrate that adenosine A1 (A1R) and A2a receptor (A2aR) agonists induce a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKY and SHR, respectively. These effects in nitrite levels are attenuated by the administration of A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR show an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolishes nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells. Finally, it is shown that the cAMP-PKA pathway is involved in A1R and A2aR-mediated decrease and increase in nitrite levels in SHR and WKY cells. Our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of neonate WKY and SHR rats. In part, the modulatory profile is different in the SHR strain.

Keywords:
Receptor knockdown Nitric oxide
Adenosine receptors
Adenosine agonists
Hypertension

1. Introduction

The nucleus tractus solitarius (NTS) is one of the main nuclei responsible for integrating signals from several brain areas or organs in order to cooperate for an orchestrated autonomic response. Previous studies have shown that many neurotransmitters and/or neuromodulators in the NTS are involved in the regulation of cardiovascular activity, including nitric oxide (NO) and adenosine. Both have cardiovascular effects strikingly similar, suggesting an interaction between adenosine and NO in the cardiovascular control in the NTS. However, the mechanisms involved in this interaction remain a controversy and may involve a complex mechanism.
Studies involving microinjection of NO donors into the NTS evokes sympathetic responses that are similar to those evoked by stimulation of the A2aR in the NTS, while the depressor and bradycardic effects of adenosine in the NTS are attenuated by a NOS inhibitor (Scislo et al., 2005). Other studies reported that NO participates in the hypotensive effect induced by A2aR stimulation (Scislo et al., 2005; Lo et al., 1998; Stella et al., 1995), and that the cardiovascular modulatory effects of adenosine in the NTS might be accomplished by NOS activation and subsequent NO release (Scislo et al., 2005; Lo et al., 1998). It is known that NO synthase exists in intrinsic neurons and in central and primary afferent terminals within the NTS (Vincent and Kimura, 1992).
On the other hand, stimulation of adenosine subtype A1R in the NTS evokes pressor responses, which were selectively blocked by DPCPX, a selective A1R antagonist (Barraco and Phillis, 1991). Studies by Scislo and O’Leary (2002) also reported that activation of adenosine A1R evokes predominantly pressor and sympathoexcitatory responses in the NTS. These have been associated to A1R-mediated inhibition of glutamate release in the NTS (Scislo and O’Leary, 2002). Jhaveri et al. (2006) showed that NO via nNOS is an endogenous regulator of the A1R expression mediated by NF-kB, suggesting an absence of a direct interaction between A1R and NO, which emphasizes the controversy that exists on this topic in the literature.
In view of this, the purpose of the present study was to analyze the modulatory action of adenosine on the nitrergic system. To accomplish this objective we have evaluated nitrite production and nNOS mRNA following treatment with the agonists and antagonists of the A1R and A2aR, as well as the analysis of the intracellular pathway involved in this modulation in cultured cells from dorsal medulla oblongata of Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Our study reports a differential modulatory effect of the A1R and A2aR on NO production, in part mediated by the nNOS isoform. It is also shown that this modulation might be mediated via the cAMP-PKA pathway. It is also clear from the data that an imbalance in the adenosine-NO interaction may be especially relevant for individuals predisposed to hypertension.

2. Material and methods

2.1. Animals

Adult male and female WKY rats and SHR from the animal housing of the Department of Physiology, Institute of Biosciences, University of Sao ˜ Paulo, Brazil, were kept in cages (in proportion 1 male to 2 female) under a regular light-dark cycle (light on at 7:00 a.m. and off at 7:00 p. m.) in temperature and humidity-controlled rooms and received food and water ad libitum. For dorsomedial medulla oblongata cell culture, 1- day-old SHR and WKY neonates were used in the present study (±12 neonates for experiment). All the procedures and protocols were performed in accordance with International and Institutional Guidelines for Animal Experimentation (protocol number 065/2008).

2.2. Cell cultures

Cell culture methodology was described in detail elsewhere (Kivell et al., 2001). Briefly, dorsomedial medulla oblongata portion was dissected out and dissociated in cold isotonic salt solution (NaCl 120 mM; KCl 5 mM; KH2PO4 1.2 mM; MgSO4.7H2O 1.2 mM; NaHCO3 25 mM and glucose 13 mM), pH 7.4. Cells were suspended in Neurobasal A media (Invitrogen) supplemented with L-glutamine (250 mol/l, Sigma), L-alanine-L-glutamine (250 mol/l, Gibco), B27 (2%, Gibco), and gentamicin (40 mg/l, Gibco). Unstained cells with Trypan Blue Stain (0.4%, Gibco) were counted and plated on poly-D-lysine-coated culture dishes (35 mm, Nunclon, USA) at the concentration of 1800 cells/mm2. The experiment was repeated three times for each strain using different pools of animal’s tissue. Cultures were kept in a humidified incubator with 5% CO2 and 95% air, at 37 ◦C, for 7 days prior to experimentation.

2.3. Treatments

Cells were treated, on the seventh day, with the selective A2aR agonist, 2-p-(2-Carboxyethyl) phenethylamino-5′-N-ethylcarboxa-midoadenosine hydrochloride hydrate, CGS 21680 (Ciba-Geigy Corporation, Sigma, USA); selective A1R agonist, CPA; A2aR antagonist 4-(2-[7- Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, ZM 241385, and A1R antagonist, CPT, (Tocris Bioscience, USA) in a concentration–response and time-course manner. Cells were treated with 0.1, 1, and 10 μM for 6, 12 and 24 h. These concentrations and times were used previously (Schulte and Fredholm, 2003). After treatment, the supernatants were used to measure the nitrite production while cells were submitted to real-time polymerase chain reaction (PCR) to evaluate nNOS mRNA levels. Inhibition of nitric oxide synthase was performed by pretreatment with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 5 μM) for 10 min followed by addition of CGS 21680 (10 μM), the A2aR agonist. The intracellular cascade involved in the effect of CGS 21680 and CPA on nitrite production was evaluated using inhibitors and activators of intracellular signaling molecules: PKA activator N6 Benzoyladenosine-3′,5′-cyclic monophosphate (6Bnz-cAMP – 5 μM – Bio Log, Germany); PKA inhibitor dihydrochloride hydrate (H89 – 5 μM – Sigma, USA) and adenylate cyclase inhibitor 9-(tetrahydro-2-furyl)adenine (SQ 22536). Drugs were added 10 min before A2aR and A1R agonist for evaluation of nitrite production. Stock solutions were prepared in dimethylsulfoxide (DMSO) or phosphate buffer sodium (PBS) and diluted in fresh culture medium immediately before use. Concentration of DMSO in the medium did not induce any changes on nitrite production.

2.4. NO production

NO formation was detected indirectly by accumulation of nitrite in culture supernatants using the DAN (diaminonaphthalene) reaction (Misko et al., 1993). Briefly, 100 μl of culture medium were incubated with 10 μl of freshly prepared DAN (0.05 mg/ml in 0.62 M HCl). After 10 min incubation, the reaction was terminated with 10 ul of 2.8 M NaOH. Formation of the 2,3-diaminonaphthotriazole was measured using fluorescent plate reader with excitation at 365 nm and emission at 450 nm. The concentration of nitrite ions was quantified using NaNO2 as standard.

2.5. Real-time PCR

Cells had total RNA extracted and purified according to illustra RNAspin protocol (Invitek, Berlim, Germany). Two-step reverse transcriptase PCR was carried out to determine the effects of A2aR and A1R agonist treatment on nNOS mRNA. High-quality total RNA was used in reverse transcription reactions to convert RNA to double-stranded cDNA using Reverse Transcription Reaction Kit (Applied Biosystems, USA). Briefly, to 1 μg of total RNA was added TaqMan buffer (1×), MgCl2 (5.5 μM), dNTPs (500 μM), random hexamers (2.5 μM), RNase inhibitor (0.4 U/μl), and the MultiScribe Reverse Transcriptase (1.25 U/μl) to a final volume of 50 μl. Two control tubes were inserted in the assay: one without the template and another without reverse transcriptase. RNA was converted into cDNA after incubations of 10 min at 25 ◦C followed by 30 min at 48 ◦C and 5 min at 95 ◦C in a thermocycler. cDNA was kept at − 80 ◦C until its use for real-time PCR. The primers, probe, and reagents for real-time PCR were commercially available at Applied Biosystems (Taqman) (Foster City, CA, USA). Evaluated nNOS mRNA was NOS1 (Taqman assay: RN00583793_m1). 18S rRNA with the reporter VIC™ (Applied Biosystems) was used as control for amplification. Data from mRNA were normalized subtracting the 18S values. Protocol provided by the manufacturer was rigorously followed.

2.6. RNA interference

For the RNA interference procedure, cultured cells of the dorsomedial medulla oblongata of WKY and SHR rats were infected by pGIPZ lentiviral vector containing shRNAmir to A2aR (VGH5518-10112715), A1R (VGM5520-99214894) and non-silencing RNAi as control (RHS4348), purchased from Open Biosystems. Briefly, after three days in culture, cells of the medulla oblongata of WKY and SHR were exposed at approximately 1 × 108 transducing Units per ml (TU/ml) for 5 h. GFP- expressing cells were observed 5 days after infection with the neurons in good condition at multiplicity of infection (m.o.i.) of 15. Three days after the infections, the RNA was extracted and immediately frozen for real time PCR analysis of A2aR (Rn00583935-m1), A1R (Rn00567668- m1) and nNOS mRNA levels.

2.7. Statistical analysis

Results were analyzed by two-way ANOVA followed by the Bonferroni post hoc test or unpaired Students t-test accessed through GraphPad Prism (GraphPad Software Inc., version 5.00, CA, USA). A p-value <0.05 was considered to indicate statistically significant differences. Data are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Nitrite modulation by adenosine receptor activation

To test whether A1R and A2aR stimulation modulates NO release in WKY and SHR cultured cells, nitrite levels was measured in response to A1R and A2aR agonist treatment. Results were evaluated at 6, 12 and 24 h after A2a (CGS21680) and A1 (CPA) agonist treatment. Considering the A2a agonist treatment, WKY cells were responsive only at the 10 μM concentration at all time period. However, SHR cells were more responsive to CGS than WKY cells, since an increase in nitrite levels was observed already at the concentration of 0.1 μM of CGS at 12 and 24 h treatment (Fig. 1A). The difference between strains, although statistically significant, was more evident at the highest concentration after 24 h treatment.
In contrast to the results observed for the A2aR, the A1R agonist, CPA, induced a significant decrease in nitrite levels in almost all concentrations and time evaluated when compared to their respective controls (Fig. 1B). At 6 h, WKY cells showed an abrupt reduction in nitrite levels at 0.1 μM while in SHR cells this concentration of CPA did not affect the nitrite levels. On the other hand, the responses to treatment with CPA at 1 μM and 10 μM were similar in WKY and SHR cells. At 12 h of treatment, the nitrite levels were significantly reduced at all concentrations tested in WKY and SHR cells when compared to control. At 24 h of treatment, both strains showed pronounced reduction in nitrite levels at 0.1 μM compared to their respective controls. After this reduction, nitrite levels were kept constant at 1 μM and 10 μM in WKY and SHR cells (Fig. 1B). Basal values observed for nitrite was usually smaller for SHR cells.

3.2. Pharmacological characterization of NO modulation by adenosine receptor activation

To further evaluate NO modulation by adenosine receptors, we have employed receptor antagonists and also activators/inhibitors of the PKA pathway. When cells were pretreated with the A2aR and A1R antagonist, ZM241385 and CPT, respectively, the effect promoted by CGS21680 and CPA was completely abolished. ZM241385 (Fig. 2A) and CPT (not shown) treatment alone did not change nitrite levels when compared to control.
To evaluate whether PKA pathway is involved in the modulation of the nitrite synthesis in medulla oblongata cultured cells, we performed pharmacological treatments to activate and inhibit PKA in the presence and absence of the CGS21680 and CPA agonists (Fig. 2). CGS21680 was used at the concentration of 10 μM for 6 h, while CPA was used at the concentration of 0.1 μM for 12 h.. Cells were treated with a PKA inhibitor (H89, 5 μM), as well as with a PKA activator (6Bnz, 5 μM). H89 was able to abolish the CGS21680 effects on nitrite levels. When PKA activator, 6Bnz, was added in the presence of CGS21680, a further increase in nitrite levels was observed compared to CGS 21680 treatment alone (Fig. 2A). On the other hand, SQ22536, an AC inhibitor, further decreased the nitrite production compared with CPA alone. The PKA activator, 6Bnz, reversed the effect induced by CPA on nitrite levels (Fig. 2B). SQ22536 and 6Bnz alone did not induce any changes in nitrite concentration (not shown).

3.3. nNOS mRNA modulation by adenosine receptor activation

The expression of nNOS mRNA was also analyzed after stimulation of A1R and A2aR. Firstly, it can be observed that the basal nNOS mRNA expression level is decreased in the SHR in comparison to the WKY rat at all periods (Fig. 3A,B). In both strains, nNOS mRNA levels exhibited a concentration-dependent increase in response to CGS 21680 after 6 h of exposure (Fig. 3A), although the increase was less expressive in cells from the SHR rat (Fig. 3A). On the other hand, we observed that after 12 h of CGS 21680 treatment, nNOS mRNA levels decreased in WKY and SHR cells when compared to respective controls (Fig. 3A). Interestingly, only WKY cells exhibited a massive increase in nNOS mRNA in response to CGS 21680 after 24 h of exposition (Fig. 3A). On the contrary, nNOS mRNA expression levels exhibited a decrease in response to CPA treatment after 6, 12 and 24 h of exposure mostly at 10 μM concentration when compared to the respective control (Fig. 3B). Strain differences are clearly seen in theses results, where SHR cells seems to be less responsive do adenosine agonists in modulate nNOS mRNA.

3.4. nNOS inhibition decreases nitrite levels

Since nNOS is found in high concentrations in many brain areas, especially in the NTS (Sapoval, 2004), we determined whether the nNOS enzyme contribute to the CGS-induced increase in nitrite levels. Using L- NAME, a non-selective nNOS inhibitor, in cultured cells from WKY and SHR, we observed a counteraction of the CGS 21680 effect on the nitrite levels in both strains (Fig. 4). L-NAME alone was not able to induce any statistical change in nitrate levels. The results also show a decreased basal nitrite levels in the SHR cells.

3.5. nNOS mRNA expression modulated by A1R and A2aR knockdown

After showing a change in the nitrergic system following stimulation of A2aR and A1R in cell culture from the dorsal brainstem of neonates WKY and SHR, we evaluate the impact of the A2aR and A1R knockdown on the nNOS mRNA expression. A2aR and A1R shRNA produced a significant knockdown in A2aR and A1R expression in cells from both strains (27.8% in WKY, 39% in SHR and 39,2% in WKY and 24,5% in SHR, respectively) (Fig. 5A,B). The A2aR knockdown was followed by a decrease in nNOS mRNA levels (22.5% in WKY and 30.9% in SHR), compared with non-silence control (Fig. 5C). On the other hand, the A1R knockdown increased the nNOS mRNA levels (29.5% in WKY and 59% in SHR), compared with non-silence control (Fig. 5D).

4. Discussion

Previous studies suggested that NO and adenosine have interrelated effects in the regulation of cardiovascular response in the NTS (Stella et al., 1995; Chen Lo et al., 1998). Our current study gives a new insight with respect to modulatory role of the A2aR and A1R on NO release taking into account nitrite production, intracellular signaling, and A1R and A2aR knockdown in cultured cells from dorsomedial medulla oblongata of neonate WKY and SHR. The most important findings in the present study are: (1) A difference in basal levels of nitrite production between the strains (2) activation of the adenosine A2aR increase the nitrite and nNOS mRNA expression levels, while activation of the A1R decrease the nitrite production and nNOS mRNA expression levels in both strains, (3) the cAMP-PKA pathway is involved in the modulation of nitrite levels by A2aR and A1R activation in WKY and SHR, (4) the nNOS isoform seems to mediate the nitrite production and (5) knockdown of A2aR and A1R increase and decrease nNOS mRNA expression, respectively.
Our results suggest that differences in the nitrergic system are already present in the neonate SHR. We have found a decreased level of nitrite and nNOS mRNA in cultured cells from dorsal medial medulla oblongata of SHR in comparison to WKY. However, earlier reports showed an up regulation of nNOS in specific brain areas of the SHR (Ferrari and Fior-Chadi, 2005; Plochocka-Zulinska and Krukoff, 1997). This might be due to the different age of rats, since we have used neonate rats and the previous studies used young pre-hypertensive rats or even old animals with established hypertension. Another difference is that we have employed cultured cells from the dorsal medial medulla oblongata and the study of Ferrari analyzed tissue sections while Plochocka- Zulinska analyzed tissue homogenate of the NTS. Results from Fig. 3 also suggest that SHR cells from the dorsomedial medulla are less responsive to adenosine receptor stimulation than WKY cells. Twenty- four hours after CGS exposure, for example, nitrite levels was massively increased in the WKY but not in the SHR, what might have important implications for development of hypertension. Also, more studies are necessary to disclose nitrergic role at the neonate rat and its involvement with the development of hypertension. Thus, this is a very good model to study cellular changes in WKY and SHR, but it is important to be aware of the differences.
We showed that the A2aR agonist treatment, CGS 21680, induced a concentration-dependent increase in nitrite levels, while administration of the A1R agonist, CPA, decreased the nitrite levels in WKY and SHR cells. These effects were significantly attenuated by the administration of the selective A2aR and A1R antagonist, ZM 241385 and CPT, respectively, which suggests that nitrite levels in both strains are modulated by adenosine A2aR and A1R. A similar result was shown in vivo by Scislo et al. (2005), which observed an increase in NO synthesis evoked by stimulation of adenosine A2aR in the NTS of rats. The interrelation between adenosine and NO is important in the cardiovascular system, including the neural control of blood pressure. The suggestion that adenosine exerts part of its cardiovascular effects by activation of A2aR, followed by stimulation of NO release in the NTS is known from the study by Chen Lo et al. (1998) and the present study details cellular mechanisms involved in this interaction.
Our study showed that activation of A1R by CPA induced a decrease in nNOS mRNA levels in both strains, while A2aR activation induced an increase in nNOS mRNA levels. It is possible that there is a dynamic mechanism between A1R and A2aR activation and nNOS mRNA levels. This mechanism can be involved in the sympathoinhibitory and sympathoexcitatory responses, via activation or inhibition of nNOS, respectively. Carvalho et al. (2006) have studied nNOS knockout mice and reported that there was a significant reduction of baroreflex responses when compared with wild-type mice. Indeed, Lin et al. (2012) using shRNA for nNOS showed that loss of nNOS expression in NTS is associated with a decreased baroreflex response. Our proposal is that an early difference in nNOS expression in SHR cells together with an imbalance between A1R and A2aR activation might collaborate with the development of high blood pressure in these animals. It is also important to point out that the nNOS mRNA modulation following A2a receptor activation by CGS21680 was almost absence at the 12 h time point. It is not possible to explain the reason of this result at this moment, but overall, the time-dependent curve of nNOS mRNA seems a U-shaped curve, which is very common in many biological models (Calabrese and Baldwin, 2001) and specifically in neurotransmitter modulation (Fior et al., 1994; Zuardi et al., 2017). It seems that the hyperstimulation in the first hours, shut the system down after 12 h of A2a receptor activation. Following this period, it is possible to see a massive increase of nNOS mRNA in the WKY, but not in the SHR, suggesting that this regulatory mechanisms in the SHR might be blunted at an early age.
We evaluated if A2aR and A1R stimulation-induced modulation in nitrite levels in cultured cells from medulla oblongata was dependent of PKA. Adenosine A1R and A2aR are coupled to distinct G protein-coupled receptors (GPCR), which have opposing effects on cyclic AMP (cAMP) production. The signaling pathways activated by A2aR are mediated via Gs protein-dependent adenylyl cyclase (AC) activation, which lead to the increase of intracellular cAMP concentration. On the other hand, the A1Rs are coupled to Gi/o proteins and inhibit AC and downstream cAMP accumulation (Burnstock, 2007; Fredholm et al., 2001). We showed that A2aR stimulation-induced increase of nitrite is a PKA-dependent mechanism whereas CGS 21680 and a PKA activator facilitated nitrite production, an effect that was inhibited by the selective PKA inhibitor, H- 89. Similar results were also showed by Rebola et al. (2002), since inhibition of PKA activity with H-89 attenuated CGS 21680 induced acetylcholine release. However, it is necessary to investigate whether a PKA-dependent mechanism acts directly activating phosphorylation or dephosphorylation of nNOS, since phosphorylation at Ser1614 (Chiang et al., 2009) and dephosphorylation at Ser847 (Xu and Krukoff, 2007) can lead to nNOS activation. We have shown that A2aR stimulation induced an increase in nNOS mRNA, which seems a PKA dependent mechanism. A similar result was obtained by Yoo et al. (2012), which demonstrated that an intracerebroventricular injection of the membrane-permeable cAMP-PKA activator, sp-cAMP, increased nNOS expression in the PVN. On the other hand, A1R exerts inhibitory effect in nitrite levels through activation of the PKA pathway, since inhibition of adenylyl cyclase by SQ22536 exacerbated the nitrite decrease and PKA activator, 6Bnz, attenuated the ability of CPA to reduce the nitrite levels.
The synthesis of NO and, consequently, of nitrite is mediated by isoforms of NOS. Considering the 3 isoforms, nNOS has a wide distribution in the brainstem (Bredt et al., 1991; Chong et al., 2019). The present data showed that the nNOS nonselective inhibitor (L-NAME) abolished the increase in nitrite levels triggered by CGS 21680 in WKY and SHR cultured cells, suggesting the involvement of nNOS isoform in this interaction. Although L-NAME inhibits both constitutive isoforms of NOS, nNOS and eNOS, the production of NO in nerve cells is catalyzed mostly by nNOS (Sapoval, 2004). Scislo et al. (2005) showed that the selective antagonist of nNOS (TRIM) and the L-NAME, abolished or reversed the depressor and sympathoinhibitory responses to A2aR stimulation in the NTS. Indeed, whereas the effects of L-NAME were only slightly stronger than those of TRIM, the hemodynamic and sympathoinhibitory responses to A2aR stimulation in the NTS are likely mediated predominantly by nNOS with a small contribution of eNOS. In light of this, based on our data it is possible to suggest a potential contribution of nNOS isoform to the nitrite levels increase evoked by A2aR stimulation in cultured cells of the dorsal medulla oblongata of WKY and SHR.
Although A2aR and A1R agonists and antagonists have provided important insights into the various functions of these receptors, the uncertainty of their selectivity can generate questions about their effects. Thus, to elucidate the function of many receptors with greater specificity, new genetic tools have emerged, such as the RNA of interference. When A2aR and A1R were knocked down in SHR and WKY cultured cells using RNA of interference, we observed a decrease of approximately 30% in the A2aR and A1R mRNA levels in cultured cells of the dorsal medulla oblongata of WKY and SHR after 5 days of infection at m.o.i. 15 using lentiviral vectors. Concomitantly, the expression levels of nNOS enzyme had an increase and a decrease of about 30% after knockdown of A1R and A2aR, respectively. Although a knockdown rate of around 25–40% is not high, Zhang et al., 2006 also found about 45% of cells expressing GFP in primary cortical cell culture 4 days post infection with a moi of 20. Our result is also consistent with a study by Cisterni et al. (2000) in which 39.7% of motoneurons were transduced using the promoter of the human cytomegalovirus (CMV). Additionally, Ralph et al. (2004) reached approximately 50% transduction efficiency using vectors based on the equine anemia virus (EIAV) for the infection of primary cultures of cortical neurons.
Studies in which the A2aR was knocked out, blood pressure and heart rate were increased and the specific A2aR agonist lost its biological activity (Ledent et al., 1997). It is also possible that the down regulation or knockout of one receptor might change the action of the other one. Since that A2aR activation is involved in the increase in nNOS mRNA expression the lack of the A1R might increase the enzyme expression, and vice e versa. This suggests an intrinsic modulation of the nitrergic system by the A2aR and A1R during the activation or reduction of receptors.
In conclusion, the present work gives a valuable contribution for understanding the action of a potent endogenous modulator such as adenosine, through its receptors, on another important modulator, the nitric oxide in cultured cells from dorsal medulla oblongata of normal and hypertensive rats. Also, our study reports a modulatory effect of A2aR activation on NO production in part mediated by nNOS isoform and performed through the cAMP-PKA pathway.

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