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CARDIOVASCULAR
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Received April 5, 2005; accepted May 10, 2005.
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Abstract |
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2-agonist -methylnorepinephrine (4 µg/rat). An increase in RVLM MAPKp42/44 occurred only after rilmenidine. Furthermore, pretreatment with efaroxan (0.15 µg/rat i.c.), a selective I1-imidazoline receptor antagonist, or with PD98059 (2'-amino-3'-methoxyflavone) (5 µg/rat i.c.), a selective extracellular signal-regulated kinase 1/2 inhibitor, significantly attenuated the hypotensive response and the elevation in RVLM MAPKp42/44 elicited by i.c. rilmenidine. The findings suggest that MAPK phosphorylation in the RVLM contributes to the hypotensive response induced by I1-receptor activation and presents in vivo evidence that distinguishes the neuronal responses triggered by the I1-receptor from those triggered by the 2-adrenergic receptor.
2-adrenergic receptor (2-AR) based on the following findings: 1) the mixed I1/2-agonist clonidine and the selective I1 agonists rilmenidine and moxonidine elicit greater hypotensive response when microinjected into the RVLM compared with the nucleus tractus solitarius (NTS); 2) the selective 2-agonist -methylnorepinephrine (-MNE) elicits appreciable hypotensive response when microinjected into the NTS (Ernsberger et al., 1995
; Ernsberger and Haxhiu, 1997); 3) imidazolinergic and adrenergic receptor signal transduction pathways seem to differ in rabbits (Chan et al., 2005); and 4) a newly selective I1-receptor agonist LNP509 reduced blood pressure, even in genetically engineered mice lacking functional 2-AR (Bruban et al., 2002; Bousquet et al., 2003).
Evidence has shown that the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) is triggered by I1-receptor in pheochromocytoma cell line (PC12) (Separovic et al., 1997; Zhang et al., 2001). The finding that microinjection of D609, a selective PC-PLC inhibitor, into the RVLM attenuated the hypotension caused by systemic moxonidine (Separovic et al., 1997) supports the existence of I1-receptor-coupled PC-PLC pathway in vivo. Furthermore, our previous findings in PC12 cells demonstrated that activation of the I1-receptor, which is coupled to PC-PLC, results in downstream activation of mitogen-activated protein kinase (MAPKp42/44) (Zhang et al., 2001). Notably, the PC12 cells, which lack 2-AR (Ernsberger et al., 1995; Separovic et al., 1996), exhibit plasma membrane imidazoline I1 binding sites (Ernsberger et al., 1995). The enhanced phosphorylation of MAPK by the selective I1-receptor agonist rilmenidine and the counteraction of such response by the selective I1-receptor antagonist efaroxan established a clear link between the I1-receptor and the MAPK signal pathway in vitro (Zhang et al., 2001). Whether enhanced phosphorylation of MAPK in vivo is essential for the mediation of the pharmacological action of selective I1 agonists has not been investigated.
The objective of the present study was to test the hypothesis that an enhanced neuronal expression of phosphorylated MAPK in the RVLM is functionally linked to the hypotension elicited by I1-receptor activation. To this end, we measured brainstem neuronal MAPKp42/44 (immunohistochemistry) and blood pressure after I1-receptor activation. We focused on the RVLM, the major site of action for the selective I1-receptor agonists such as rilmenidine and moxonidine (Gomez et al., 1991; Haxhiu et al., 1994). However, since rilmenidine exhibits 2-AR agonist activity (Szabo et al., 1993; Regunathan et al., 1995; Zhu et al., 1999) and the RVLM contains both I1- and 2-ARs (Reis, 1996), we investigated the effects of the pure 2-AR agonist -MNE on MAPK phosphorylation in the RVLM. As a control, we investigated whether rilmenidine enhanced the phosphorylation of MAPK in the NTS, which is devoid of functional I1-receptor (Gomez et al., 1991). To confirm the involvement of the I1-receptor and the MAPK pathway in the observed responses, we investigated the effects of the selective I1-receptor antagonist efaroxan and the ERK1/2 inhibitor PD98059 on the hypotensive response and the enhanced RVLM MAPKp42/44 elicited by rilmenidine.
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Materials and Methods |
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Intracisternal Cannulation. Four to 6 days before starting the experiment, we implanted a stainless steel guide cannula into the cisterna magna under pentobarbital anesthesia (50 mg/kg i.p.) as in our previous studies (El-Mas et al., 1994a; El-Mas and Abdel-Rahman, 1998). Briefly, the steel cannula (23-gauge; Small Parts, Miami Lakes, FL) was passed between the occipital bone and the cerebellum so that its tip protruded into the cisterna magna. The cannula was secured by dental acrylic cement (Duralon; Thomas Dental Supply, Raleigh, NC) as described in our previous studies. The guide cannula was considered patent when spontaneous outflow of cerebrospinal fluid was observed and by gross post-mortem histological verification after perfusion with fixation solution. Each rat received a subcutaneous injection of the analgesic buprenorphine hydrochloride (Buprenex; 0.3 µg/rat) and an intramuscular injection of 60,000 U of penicillin G benzathine and penicillin G procaine in aqueous suspension (Durapen). After intracisternal cannulation, the rats were housed individually.
Intravascular Cannulation. For measurement of blood pressure (BP) and heart rate (HR), the method described in our previous studies (El-Mas et al., 1994b; El-Mas and Abdel-Rahman, 1998) was adopted. In brief, the rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.). Catheters (polyethylene-50) were placed in the abdominal aorta and vena cava via the femoral artery and vein for measurement of BP and i.v. administration of drugs, respectively. The catheters were inserted 
5 cm into the femoral vessels and secured in place with sutures. The catheters were flushed with heparin (200 U/ml). The arterial catheter was connected to a pressure transducer (Gould-Statham, Oxnard, CA), and BP was displayed on Grass polygraph (model 7D; Grass Instruments, Quincy, MA). HR was computed from BP waveforms by a Grass tachograph and was displayed on another channel of the polygraph.
Immunohistochemistry. The procedure reported in Current Protocols in Neuroscience for immunohistochemistry for light microscopy (Gerfen, 1997; Ince and Levey, 1997) was followed. Briefly, brain fixation was achieved by trans-cardiac perfusion of the fixation solution (4% paraformaldehyde) after a lethal dose of pentobarbital. Then, the brain was transferred into 30% sucrose in Tris-buffered saline (TBS) for infiltration until the brain sinks. The brain was cut serially at –22°C with a microtome cryostat (HM 505 E; Microm International GmbH, Walldorf, Germany). Six to eight sections (16 µm in thickness) of the brain were collected in each well of a cell culture plate (12 wells; BD Biosciences, San Jose, CA) containing ice-cold TBS. Before the immunostaining procedure, a section from each well was stained with thionin (Gerfen, 1997) for the segment mapping according to Paxinos and Watson (1982). The sections from the coordinated segments (–12.8 to approximately –11.80 mm) were used for immunostaining (Fig. 2). ABC method was used following the manufacturer's instruction (Vectastain ABC kits; Vector Laboratories, Burlingame, CA) with minor modification (Ince and Levey, 1997). The sections from treatment and control groups were simultaneously incubated with the primary antibody, anti-phospho-MAPK (p42/44) monoclonal antibody (Cell Signaling Technology Inc., Beverly, MA) (1:400 dilution). After rinsing with TBS, H2O2/3.3'-diaminobenzidine solution was added and the sections were examined under a microscope (Nikon Diaphot 300; Nikon, Tokyo, Japan) for the appearance of reddish brown staining. After dehydrating and clearing, the sections were sealed with Permount (Fisher Scientific Co., Pittsburgh, PA) and observed under the microscope. The images were processed by Micropublisher (QImaging, Burnaby, BC, Canada). For collecting data of the phosphorylated MAPK, the cell body with dark brown immunoreactive product was considered a positive cell. The positive cells in the defined RVLM or NTS of the segments from –12.8 to –11.8 mm according to the rat brain atlas (Paxinos and Watson, 1982) were counted on each side of the RVLM or NTS from the six sections of similar segment of each rat. The data of the positive cell count from the RVLM represented the "numbers/side/rat". The average of the positive cell number from each rat was used for the statistical analysis.
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-MNE (4 µg/rat; Sigma-Aldrich, St. Louis, MO), or equal volume (4 µl) of artificial cerebrospinal fluid (ACSF: 123 mM NaCl, 0.86 mM CaCl2, 3 mM KCl, 0.89 mM MgCl2, 25 mM NaHCO3, 0.5 mM NaH2PO4, and 0.25 mM Na2HPO4, pH 7.4). In the third experiment, efaroxan (0.15 µg/rat i.c.; Sigma-Aldrich) or equal volume of ACSF was administrated and followed 10 min later with i.c. rilmenidine (25 µg/rat). In the fourth experiment, the rats received the ERK1/2 inhibitor PD98059 (5 µg/rat; Sigma-Aldrich) or equal volume of vehicle (DMSO) i.c. 10 min before rilmenidine (25 µg/rat i.c.). In all experiments, the blood pressure and heart rate were recorded for additional 15 min after rilmenidine or its vehicle (ACSF) after which each rat was prepared for trans-cardiac perfusion and brain preparation for immunohistochemical detection of phosphorylated MAPK as detailed above.
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Statistical Analysis. Values are presented as mean ± S.E.M. Mean arterial pressure (MAP) was calculated as diastolic + [(systolic–diastolic)/3]. Statistical comparison was made by analysis of variance followed by post hoc multiple comparisons of the mean with Tukey's test. Student's t test (unpaired, two-tailed) was used for comparing the mean of the baseline and positive cell count data. The probability levels less than 0.05 were considered as statistically significant.
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Results |
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). Also, in the immunohistochemical studies, a negative control (omitting the primary antibody) was run, which resulted in a clean background (data not shown). The specific immunostaining of cells was mostly distributed in the RVLM (Fig. 1). Both the cell body and the processes of the neuron were stained. Some of the cerebral blood vessels were stained nonspecifically, but they are not included in the data analysis. For comparison of the level of expression of phosphorylated MAPK, all brain sections were processed similarly, and the number of cells expressing the phosphorylated MAPK in the RVLM was counted in similar segments of the sections obtained from treatment and control rats. Possible immunostaining for MAPKP42/44 in the neurons of the NTS in the same section was also evaluated. The sections from the desired segment of the RVLM were verified by thionin staining before performing the immunohistochemical studies. Figure 1, A and B, shows a representative example of phosphorylated MAPK immunostaining (Fig. 1B) and the verification of the site by thionin staining (Fig. 1A) after i.v. rilmenidine
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Effect of Central Intracisternal Administration of Rilmenidine or -MNE on Blood Pressure and MAPK Phosphorylation in the RVLM. To verify whether the phosphorylation of MAPK in the RVLM was elicited by the I1-imidazoline receptor or the 2-adrenergic receptor, rilmenidine (25 µg/rat) or the pure 2-adrenergic receptor agonist -MNE (4 µg/rat), in doses selected in anticipation of causing equal hypotensive responses, was injected into the cisterna magna. Rilmenidine and -MNE elicited similar hypotensive responses, whereas ACSF had no effect on blood pressure or heart rate (Fig. 2). However, only rilmenidine caused a significant increase in the expression of phosphorylated MAPK in RVLM (Fig. 3). The number of the positive cells expressing phosphorylated MAPK in RVLM was markedly increased (p < 0.05) by rilmenidine (19 ± 1) compared with ACSF (7 ± 1). Compared with ACSF, -MNE had no effect on neuronal expression of MAPKP42/44 in the RVLM (Fig. 3). Neither rilmenidine nor -MNE increased MAPK phosphorylation in the NTS (Fig. 3).
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), was used in this experiment. Intracisternal efaroxan (0.15 µg/rat), which had no effect on blood pressure or heart rate, attenuated the hypotensive and bradycardic responses elicited by subsequent i.c. rilmenidine (25 µg/rat; Fig. 4). Compared with ACSF, efaroxan significantly (p < 0.05) attenuated the hypotensive (–10.0 ± 1.9 versus –20.6 ± 4.5 mm Hg) and bradycardic (–49 ± 9 versus –113 ± 15 beats/min) responses and substantially (p < 0.05) reduced the number of positive cells, which expressed phosphorylated MAPK (P42/44) in the RVLM (7 ± 1 versus 16 + 2) elicited by i.c. rilmenidine (Fig. 5).
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), was used in this experiment. Intracisternal PD98059 had no significant effect on blood pressure (–2.1 ± 3.3 mm Hg) or heart rate (–14 ± 17 beats/min) during the observation period (Fig. 6). Compared with the vehicle (DMSO), pretreatment with PD98059 virtually abolished the hypotensive response (Fig. 6) and the enhancement of MAPK phosphorylation (MAPKP42/44) in the RVLM neurons (Fig. 7) caused by i.c. rilmenidine.
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Discussion |
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2-agonist -MNE; and 3) the selective I1-receptor antagonist efaroxan or the ERK1/2 inhibitor PD98059 significantly attenuated the hypotensive response and virtually abolished the enhancement of neuronal MAPK phosphorylation elicited by rilmenidine. Together, these findings suggest a functional role for RVLM neuronal MAPKp42/44 in the hypotension elicited by the I1-receptor activation and present evidence that highlights a neuronal mechanism for the I1-receptor that is distinct from the signal transduction pathway triggered by the 2-AR.
Results of the present study demonstrated that rilmenidine-evoked hypotension was associated with significant enhancement of MAPK phosphorylation in the RVLM neurons. This finding extends our previous findings in differentiated PC12 cells, which demonstrated concentration-dependent enhancement of MAPK phosphorylation by rilmenidine (Zhang et al., 2001). The finding was later confirmed by Edwards et al. (2001). More importantly, the current findings highlight the interesting possibility that the enhanced phosphorylation of MAPK in the RVLM, triggered by the I1-receptor activation, represents a novel neuronal mechanism for the hypotensive action of the second generation, centrally acting drugs such as rilmenidine and moxonidine. The present findings may explain, at least partly, the very recently reported difference in the neural pathways that mediate the hypotensive actions of clonidine and moxonidine (Chan et al., 2005).
We measured phosphorylated MAPK as intracellular signal that reflects the interaction between the I1-receptor and PC-PLC. Reported studies, including ours, have delineated this signal transduction pathway in differentiated (nerve growth factor-treated) PC12 cells (Separovic et al., 1997; Zhang et al., 2001). Notably, the present study is the first to replicate such a pathway in vivo and circumvents some of the limitations of the cell culture studies such as the required addition of nerve growth factor to the culture medium (Separovic et al., 1997; Zhang et al., 2001). It was imperative that we observe enhanced phosphorylation of MAPK in the RVLM neurons if such neuronal signaling bears relevance to the pharmacological (hypotensive) action produced by I1-receptor activation. This is because the RVLM is the major site for the I1-mediated hypotension (Haxhiu et al., 1994; Ernsberger et al., 1995; Ernsberger and Haxhiu, 1997; Head et al., 1998). Notably, the findings of the first set of experiments in the present study provided correlative association between the enhanced neuronal MAPK phosphorylation in the RVLM and the hypotensive response elicited by systemic rilmenidine. Furthermore, it may be argued that the neuronal signal and/or the hypotensive response resulted, at least in part, from the activation of the 2-AR because 1) the RVLM contains I1- and 2-ARs (Reis, 1996); and 2) rilmenidine, although a selective I1 agonist, also activates the 2-AR (Szabo, 2002). In effect, some investigators suggest that the hypotensive action of the second generation, centrally acting drugs depends, directly or indirectly, on the 2-AR (Szabo et al., 1993; Regunathan et al., 1995; Zhu et al., 1999).
To rule out a possible involvement of the 2-AR in the enhanced MAPK phosphorylation in the RVLM caused by rilmenidine, we administered intracisternally -MNE, which is considered pure 2-receptor agonist (Szabo, 2002). Because -MNE failed to enhance the phosphorylation of MAPK in the RVLM, while producing a hypotensive response similar to that elicited by rilmenidine, our findings rule out the involvement of 2-AR in the modulation MAPK phosphorylation in the RVLM. It is also important to note that the following findings rule out the possibility that the enhanced phosphorylation of MAPK in the RVLM was caused by changes in blood pressure. First, the enhancement of RVLM MAPK phosphorylation after i.c. rilmenidine rules out the possibility that the initial pressor response or other peripheral effects caused by i.v. rilmenidine in our preliminary study (Fig. 1) contributed to this response. Second, a reduction in blood pressure elicited by -MNE, similar to that elicited by rilmenidine, was not associated with enhanced phosphorylation of MAPK in the RVLM. Together, these findings suggest that the enhanced phosphorylation of MAPK in the RVLM is mediated via the activation of central I1-receptor.
It must be remembered that we report significant increase in RVLM neuronal MAPK phosphorylation (P42/44) at one time point, 15 min after drug administration. This time was chosen because our previous findings in PC12 cells showed MAPK phosphorylation starts at 2 to 5 min and reaches a maximal response 15 min after I1-receptor activation (Zhang et al., 2001). Nonetheless, the possibility must be considered that the enhanced phosphorylation of MAPK in the RVLM and the hypotensive response caused by rilmenidine may be unrelated events. Notably, previous attempts to link the PC-PLC signal transduction pathway to the hypotensive response elicited by another I1-receptor agonist, moxonidine, were confounded by some experimental conditions. Separovic et al. (1997) showed that pretreatment with the PC-PLC inhibitor D609 abolished the hypotensive effect of moxonidine. However, the significant pressor response caused by intra-RVLM D609 in the reported study may have confounded the data interpretation. Furthermore, the specificity of D609 to PC-PLC, which has been questioned in cell culture studies, including ours (Zhang et al., 2001), has not been evaluated in vivo. No neuronal marker that reflects the activation of the PC-PLC in the absence and presence of D609 was investigated in the reported in vivo study (Separovic et al., 1997). In the present study, the selective I1 antagonist efaroxan, used in a dose that preferentially blocks the I1-receptor (El-Mas and Abdel-Rahman, 2001), virtually abolished the enhancement in MAPK phosphorylation in RVLM neurons and the hypotensive response elicited by I1-receptor activation.
Furthermore, we reasoned that if the enhanced phosphorylation of MAPK in the RVLM plays a causal role, at least partly, in the I1-mediated hypotension, then inhibition of MAPK phosphorylation is expected to attenuate the hypotensive response. To achieve this goal, we used PD98059, a selective inhibitor of the activation of MAPK kinase, an upstream regulator of MAPK (Alessi et al., 1995), in a dose that exhibits no neurotoxic effect in vivo (Seyedabadi et al., 2001). In support of our hypothesis, PD98059 virtually abolished the enhancement of MAPK phosphorylation (MAPKP42/44) in the RVLM and the hypotensive response caused by rilmenidine. These results support a possible causal role for phosphorylated MAPKP42/44 in the RVLM in the hypotensive action of rilmenidine. However, a contradictory role for MAPKp42/44 in the RVLM in blood pressure regulation has been reported. In anesthetized spontaneously hypertensive rats and Wistar-Kyoto rats, intra-RVLM PD98059 lowered blood pressure, which has been attributed to inhibition of PD98059-sensitive genomic processes (Seyedabadi et al., 2001). The objective of the reported study was to allow time (14 h) for possible transcriptional and translational consequences to take place after kinase inhibition (Seyedabadi et al., 2001). We focused on the rapid effects of PD98059 because the I1-mediated phosphorylation of MAPK starts within minutes and peaks at 15 min (Zhang et al., 2001). Nonetheless, in spite of the differences in experimental conditions between the present and the reported (Seyedabadi et al., 2001) studies, the blood pressure remained virtually unaltered during the first 30 to 60 min after i.c. injection (present study) or intra-RVLM (Seyedabadi et al., 2001) injection of PD98059. Whether the delayed hypotensive response observed after intra-RVLM PD98059 was related to changes in RVLM MAPKp42/44 is not known because the latter was not measured in the reported study (Seyedabadi et al., 2001).
In conclusion, the present findings suggest a causal relationship between enhanced phosphorylation of MAPK in the RVLM neurons and the hypotensive response elicited by the central I1-receptor activation. The enhanced phosphorylation of MAPK serves as a reliable marker for the PC-PLC signal transduction pathway triggered by I1-receptor activation in cell culture as well as in vivo, which establishes relevance for this pathway to the hypotensive response elicited by the second generation, centrally acting drugs such as rilmenidine. The present findings provide the first evidence that yields insight into the consequence of the rapid phosphorylation of MAPKP42/44 in the RVLM on blood pressure and supports a unique neuronal signaling for the I1-receptor in vivo.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: 2-AR, 2-adrenergic receptor; RVLM, rostral ventrolateral medulla; NTS, nucleus tractus solitarius; -MNE, -methylnorepinephrine; PC-PLC, phosphatidylcholine-specific phospholipase C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PD98059, 2'-amino-3'-methoxyflavone; BP, blood pressure; HR, heart rate; TBS, Tris-buffered saline; ACSF, artificial cerebrospinal fluid; i.c., intracisternal; DMSO, dimethyl sulfoxide; MAP, mean arterial pressure; D609, tricyclodecan-9-yl-xanthogenate.
Address correspondence to: Dr. Abdel A. Abdel-Rahman, Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27834. E-mail: abdelrahmana{at}mail.ecu.edu
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