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CARDIOVASCULAR
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
Received December 28, 2007; accepted April 23, 2008.
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Abstract |
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-nitro-L-arginine methyl ester into the lateral cerebral ventricle via osmotic minipump for 7 days. We found that 1) in CHF rabbits, intracerebroventricular infusion of SIM significantly suppressed basal RSNA (1st day 69.5 ± 8.9% maximum; 7th day 26.0 ± 6.0% maximum; P < 0.05, n = 7) and enhanced arterial baroreflex function starting from the 2nd day and lasting through the following 5 days; 2) statin treatment significantly up-regulated neuronal nitric-oxide synthase (nNOS) protein expression in the rostral ventrolateral medulla (RVLM) (control, n = 6, 0.12 ± 0.04; SIM-treated, n = 7, 0.31 ± 0.05. P < 0.05); 3) in CATH.a neurons, incubation with SIM significantly up-regulated the nNOS mRNA expression, which was blocked by coincubation with mevalonate, farnesyl-pyrophosphate, or geranylgeranyl-pyrophosphate; and 4) incubation with Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] significantly up-regulated nNOS mRNA expression in these neurons. These results suggest that central treatment with SIM decreased sympathetic outflow in CHF rabbits via up-regulation of nNOS expression in RVLM, which may be due to the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and a decrease in Rho kinase by SIM.
) and blunted arterial baroreflex function (DiBona and Sawin, 1995). Our previous studies demonstrated that oral treatment with the HMG-CoA reductase inhibitor, simvastatin (SIM), improved baroreflex function and normalized sympathetic outflow in the rabbits with CHF (Pliquett et al., 2003). In previous experiments, we have demonstrated that these therapeutic effects of SIM correlated with changes in several important signaling molecules in the rostral ventrolateral medulla (RVLM) of rabbits with CHF(Gao et al., 2005a). These experiments were based on systemic (oral) administration of SIM. Therefore, it is difficult to determine whether these effects were mediated by central or peripheral mechanisms following oral administration of SIM. Because SIM has been demonstrated to permeate the blood-brain barrier (Saheki et al., 1994), we postulated that the above effects of oral SIM were mediated, at least partially, by central mechanisms. Our first hypothesis in the current experiment was that direct administration of SIM into the brain would reduce sympathetic nerve activity in the CHF state.
There is considerable evidence suggesting that nitric oxide (NO) in the central nervous system, especially in the brain stem, plays an important role in the regulation of sympathetic outflow and blood pressure (Zanzinger, 1999; Krukoff, 1999). The RVLM is the last relay station in the brain to integrate sympathetic outflow (Dampney, 1994). The sympathetic premotor neurons of the RVLM provide the major tonic excitatory input to sympathetic preganglionic neurons in the spinal cord (Guertzenstein and Silver, 1974). Chan et al. (2001, 2003) have demonstrated the existence of nitric-oxide synthase (NOS) in the RVLM. On the other hand, Bredt et al. (1990) demonstrated that NO synthase in the brain is exclusively associated with discrete neuronal populations. Treatment with a precursor of NO (Shapoval et al., 1991) or an NO donor (Kagiyama et al., 1997) into the RVLM caused a marked depressor response. In contrast, NOS inhibitors were found to cause a pressor response in anesthetized animals (Tseng et al., 1996).
It is interesting that Hirooka et al. (2003) reported that reduced nNOS expression in the RVLM and that the resulting reduction of NO production contributed to the enhanced sympathetic drive in CHF rats. Using the same animal model, we found that delivery of Ad.nNOS into the RVLM to increase nNOS expression and NO production normalized sympathetic outflow and enhanced arterial baroreflex function (Wang et al., 2003). In spontaneously hypertensive rats, another animal model characterized by sympatho-excitation, increased NO production caused by the overexpression of eNOS in the RVLM led to an increase in the maximal gain of the baroreflex control of heart rate (HR) (Kishi et al., 2003) and a decrease in mean arterial pressure (MAP), HR, sympathetic nerve activity, and 24-h urinary norepinephrine excretion (Kishi et al., 2002).
On the other hand, recent animal experiments have demonstrated the up-regulation of activity and expression for inducible NOS (Ye et al., 2006), eNOS (Di Napoli et al., 2001), and nNOS (Nakata et al., 2007) by statins. Therefore, our second hypothesis in the current experiments is that SIM in the rabbit with CHF normalizes sympathetic outflow via a central nNOS-NO mechanism. The primary goals of this experiment were to determine the effect of chronic intracerebroventricular infusion of SIM on renal sympathetic nerve activity (RSNA), arterial baroreflex function, and nNOS protein expression in the RVLM of CHF rabbits. In addition, we also explored the pathway mediating the SIM-induced nNOS expression using a neuronal cell culture.
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Materials and Methods |
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Induction of CHF. CHF was induced by chronic ventricular tachycardia for 3 to 4 weeks, as described previously (Pliquett et al., 2003). Heart failure was characterized by a minimum of a 50% reduction in baseline ejection fraction compared with the pre-paced state, a 2-mm dilation of the left ventricle in both systole and diastole, and by clinical signs of CHF, such as pleural fluid, ascites, pulmonary congestion, and cachexia.
Chronic Lateral Cerebral Ventricle Infusion. All rabbits received an implantation of a 19-gauge cannula into the lateral cerebral ventricle as described previously (Gao et al., 2005b). An osmotic mini pump (model 2001; Durect Corporation, Cupertino, CA) filled with artificial CSF, SIM (5 µg/0.5 µl/h), or SIM (5 µg/0.5 µl/h) + L-NAME (50 µg/0.5 µl/h) was implanted subcutaneously in the back of neck and connected to the cerebroventricular cannula. At the end of each experiment, the placement of the cannula was confirmed by injection of 50 µl of 2% Pontamine Sky Blue into the lateral cerebral ventricle via the cannula.
Arterial Pressure, Heart Rate, and Renal Sympathetic Nerve Recording. A catheter connected to a radiotelemetry unit (Data Sciences International, St. Paul, MN) was inserted into the descending aorta via a branch of the right femoral artery for direct measurement of arterial pressure (AP). HR was derived from the AP pulse using a PowerLab (model 8S; AD Instruments Inc., Colorado Springs, CO) data acquisition system. Renal sympathetic nerve recording was carried out in the conscious state as described previously (Liu and Zucker, 1999).
Evaluation of Arterial Baroreflex Function. AP, HR, and RSNA were recorded on a Powerlab system. An intravenous infusion of sodium nitroprusside and then phenylephrine was used to induce alterations of AP. Baroreflex sensitivity was expressed as the slope of the linear regression relating changes in integrative RSNA to changes in MAP from the lowest AP induced by nitroprusside to the peak pressure induced by phenylephrine.
Preparation of RVLM Tissue. At the end of experiment, the rabbits were euthanized with pentobarbital sodium. The brain was removed and immediately frozen on dry ice, blocked in the coronal plane, and sectioned at 150-µm thickness in a cryostat. The bilateral RVLM areas (2.0–3.5 mm from the obex, 2.5–4.0 mm from middle line, and within the ventrolateral area of section) were punched out using a 15-gauge needle stub (inner diameter of 1.5 mm) for the analysis of protein of nNOS.
Western Blot Analysis for nNOS. Protein from RVLM of rabbits was extracted using radioimmunoprecipitation assay buffer, the concentration of which was then measured using a protein assay kit (Pierce, Rockford, IL) and adjusted to the same with equal volumes of 2 x 4% SDS sample buffer. The samples were then boiled for 5 min following by loading on the 7.5% SDS-polyacrylamide gel electrophoresis gel (5 µg of protein/30 µl/well) for electrophoresis using Bio-Rad mini gel apparatus (Bio-Rad, Hercules, CA) at 40 mA/each gel for 45 min. The fractionized proteins on the gel then were electrophoretically transferred onto the polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA) at 300 mA for 90 min. The membrane was probed with primary antibody (nNOS mouse lgG2a, 1:1000; BD Biosciences, San Jose, CA) and secondary antibody (goat anti-mouse IgG-HRP, 1:2500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then treated with enhanced chemiluminescence substrate (Pierce) for 5 min at room temperature. The bands in the membrane were visualized and analyzed using UVP BioImaging Systems (Upland, CA).
CATH.a Cell Culture. A neuronal cell line (CATH.a) was purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 containing 8% horse serum, 4% fetal bovine serum, and 1% penicillin-streptomycin at 37°C in a humidified atmosphere equilibrated with 5% CO2. After subculture, cells were plated on polystyrene tissue culture dishes at a density of 1 x 107 cells/100-mm plate with N-6,2'-O-dibutyryladenosine 3',5'-cycle-monophosphate (1 mM; Sigma-Aldrich, St. Louis, MO) to grow for 2 days to obtain differentiated CATH.a cells and then were treated with SIM alone or plus mevalonate, farnesylpyrophosphate (FPP), geranylgeranylpyrophosphate (GGPP), or Y-27632 for 1 to 3 days.
Real-time RT-PCR Analysis of nNOS mRNA. Total RNA from CATH.a cell pellets for real-time RT-PCR was extracted using RNeasy columns (Qiagen, Valencia, CA), which then was reverse-transcribed into double-stranded cDNA. Real-time RT-PCR was carried out using the thermocycler (PTC-200 Peltier Thermal Cycler with CHROMO 4 Continuous Fluorescence Detector; Bio-Rad) according to the manufacturer's recommendations. Cycle numbers obtained at the log-linear phase of the reaction were plotted against a standard curve prepared with serially diluted control samples. Expression of target genes was normalized by GAPDH levels. The primers and probes used in this experiment were designed using software on the website (https://www.genscript.com/ssl-bin/app/primer) and synthesized in the Eppley Institute Molecular Biology Core Laboratory, University of Nebraska Medical Center. Table 1 shows the gene-specific primers and probes. The primers and probes to detect gene expression in the CATH.a neuron sample were designed according to the mouse gene sequences because these neurons are derived from a transgenic mouse brain locus coeruleus.
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Statistical Analysis. Data are expressed as the mean ± S.E. The differences between groups were determined with a one-way analysis of variance followed by the Student's Newman-Keuls test for analysis of significance. The differences before and after intracerebroventricular infusion in each group were analyzed with a paired t test. Statistical significance was defined as P < 0.05.
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Results |
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). There were no significant changes in these parameters between the three groups of rabbits studied here.
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Intracerebroventricular Infusion of SIM Decreases Baseline RSNA. An original recording of AP, HR, and RSNA pre- and 7 days postintracerebroventricular infusion of SIM in conscious CHF rabbits is shown in Fig. 1. Compared with Fig. 1A (before infusion), intracerebroventricular infusion of vehicle (artificial CSF) did not exhibit any effects on the RSNA (Fig. 1B). In contrast, from Fig. 1D, we can clearly see a noticeable decrease in basal RSNA at day 7 after intracerebroventricular infusion of SIM compared with the pre-SIM infusion (Fig. 1C), indicating that central treatment with SIM suppressed sympathetic nerve activity in the CHF state. However, we did not see a marked alteration of blood pressure or heart rate after intracerebroventricular infusion of vehicle, simvastatin, or simvastatin plus L-NAME. Figure 2 is the grouped data illustrating the effects of intracerebroventricular infusion of SIM on basal RSNA in conscious CHF rabbits. From this figure, we can see that intracerebroventricular infusion of SIM caused a decrease in RSNA beginning at day 2, which was sustained up to day 7, compared with either day 0 or vehicle treatment. On the other hand, intracerebroventricular infusion of SIM plus L-NAME prevented the sympatho-inhibition of SIM alone. This suggests that the response to SIM was, at least in part, due to an NO mechanism.
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It is worthy to note that we also measured nNOS protein expression in the hypothalamus, another sympathetic-related nucleus, in these rabbits. We found higher expression of nNOS protein in this area, but we did not find significant differences between vehicle, SIM, and SIM plus L-NAME-treated rabbits. On the other hand, in two CHF rabbits with intravenous infusion of the same dose of SIM (5 µg/0.5 µl/h for 7 days), we did not find alterations of baseline RSNA, baroreflex function, and nNOS protein expression in the RVLM (data not shown).
RhoA/ROCK Pathway Mediates the SIM-Induced Up-Regulation of nNOS Expression. In this experiment, we employed the CATH.a neuronal cell line to explore the mechanisms mediating the effect of SIM on nNOS expression. We first observed the effects of three doses of SIM (0.1, 1, and 10 µM) on the nNOS mRNA expression. We found that 0.1 and 1 µM SIM significantly up-regulated nNOS mRNA expression; however, 10 mM SIM had no effect compared with the vehicle treatment (nNOS/GAPDH: 0.20 ± 0.09 in control, 0.52 ± 0.11 at 0.1 µM, 0.59 ± 0.12 at 1 µM, and 0.18 ± 0.07 in 10 µM SIM; P < 0.05). We also determined the time course of 1 µM SIM-induced nNOS mRNA expression, demonstrating that 48, 72, and 96 h of treatment of SIM significantly up-regulated nNOS mRNA expression and that 24 h of treatment only exhibited a tendency to increase the nNOS mRNA expression but did not reach the statistical significance compared with control (nNOS/GAPDH: 0.10 ± 0.09 in control, 0.38 ± 0.11 in 24 h, 0.71 ± 0.14 in 48 h, 0.52 ± 0.10 in 48 h, and 0.47 ± 0.14 in 96 h SIM treatment; P < 0.05).
Figure 6 shows the effects of the HMG-CoA reductase products on the SIM-induced up-regulation of nNOS mRNA expression in CATH.a neurons. SIM (1 µM) incubation for 48 h significantly up-regulated nNOS mRNA expression, which was completely abolished by the L-mevalonate (200 µm), FPP (5 µm), and GGPP (5 µm). On the other hand, the inhibitor of Rho-associated kinase (ROCK), Y-27632 (100 µm), significantly up-regulated nNOS mRNA expression.
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Discussion |
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; Gao et al., 2005a). In the current study, we further found that direct intracerebroventricular infusion of SIM also exhibited a beneficial effect on sympathetic nerve activity and baroreflex function in the CHF state. This was accompanied by an up-regulation of nNOS protein expression in the RVLM. Moreover, intracerebroventricular coinfusion of SIM and L-NAME completely abolished the effect of SIM on sympathetic nerve activity and baroreflex function but had no effects on nNOS expression. These results strongly suggest that increased central NO production is another critical pathway to normalize sympathetic nerve activity in the CHF state and that the elevated NO production at least partially resulted from the up-regulation of nNOS expression in the RVLM.
As indicated above, NOS and NO in the RVLM have a major influence on sympathetic nerve activity in both physiological and pathological states. Vincent and Kimura (1992) first demonstrated the presence of NOS-immunoreactive neurons in the RVLM of rats, which was further confirmed by Ohta et al. (1993) and Simonian and Herbison (1996). This morphological evidence implies a potentially functional involvement of RVLM local NO in the regulation of sympathetic outflow. Indeed, microinjection of sodium nitroprusside, a donor of NO, or L-arginine, a precursor for NO, into the RVLM of normal cats induced a reduction in RSNA and MAP (Shapoval et al., 1991). In anesthetized rats, microinjections of NO donors and NOS inhibitors into the RVLM exhibited sympatho-inhibitory and sympatho-excitatory effects, respectively (Zanzinger et al., 1995). In the current experiment, we found that intracerebroventricular infusion of SIM up-regulated nNOS protein expression in the RVLM of CHF rabbits and the simultaneously decreased RSNA, suggesting that SIM-induced suppression of sympathetic outflow was mediated, at least partially, by the NO/nNOS pathway. Indeed, the NOS inhibitor, L-NAME, abolished the effects of SIM on sympathetic nerve activity when coinfused with SIM, providing further evidence demonstrating the potential involvement of NO in this SIM effect. Moreover, functional data from this experiment show that the SIM-induced decrease in sympathetic nerve activity presented as early as day 2 after treatment and was sustained up to day 7 after SIM treatment. This was paralleled by an up-regulated nNOS protein expression at days 2 and 7 after SIM treatment. Recently, Kishi et al. (2001) reported that gene transfer-induced overexpression of eNOS in the bilateral RVLM significantly decreased AP, HR, and sympathetic nerve activity in conscious rats, providing a more direct involvement of the RVLM NOS expression in the regulation of sympathetic outflow. They further demonstrated that eNOS overexpression-induced inhibition of sympathetic activity was mediated by an increased release of GABA in the RVLM. However, using antibodies from BD Biosciences, we did not detect the NOS and eNOS protein expressions in the RVLM of rabbits in this current experiment (data not shown).
Another novel finding in this experiment is that intracerebroventricular infusion of SIM improved arterial baroreflex function of CHF rabbits. This may also be mediated by the NO/nNOS pathway in the RVLM because it was reversed by L-NAME treatment. Indeed, overexpression of eNOS or nNOS by gene transfer into the RVLM has been demonstrated to improve the impaired arterial baroreflex function in either spontaneously hypertensive rats (Kishi et al., 2003) or CHF rats (Wang et al., 2003). Therefore, we postulated that SIM would suppress sympathetic nerve activity and the improved arterial baroreflex function. Based on the known effects of statins, we hypothesized that both of these effects would be due to up-regulation of nNOS expression in the RVLM. It is well documented that sympathetic nerve activity and arterial baroreflex function are tightly interdependent. That is, activation of the arterial baroreflex markedly inhibits sympathetic outflow, and sympathetic overactivity impairs baroreflex function. However based on the current experiments, it is difficult to determine whether these are two independent processes after treatment with SIM in CHF rabbits. Comparing the data in Figs. 2 and 4, we noted that the slope of the baroreflex was gradually increased from day 1 to day 7 after SIM treatment, with the peak at day 7. However, the basal RSNA appears to be decreased to the same degree from day 3 to day 7 after SIM treatment. Moreover, at days 2 and 3, the basal RSNA in the SIM-treated rabbits was lower than both before SIM treatment and vehicle treatment of rabbits (Fig. 2); however, this was not the case for the arterial baroreflex (Fig. 4). Therefore, we tend to regard the SIM-induced inhibition of sympathetic activity as the cause of the SIM-induced improvement in baroreflex function. Based on these data, we believe that the critical sequences of events after SIM treatment are as follows. SIM up-regulates nNOS expression in the RVLM and then suppresses sympathetic nerve activity, which in turn improves arterial baroreflex function.
One critical question raised from the above results is how SIM up-regulates nNOS expression. Statins, including SIM, are potent inhibitors of HMG-CoA reductase and cholesterol biosynthesis and, therefore, are widely used in the treatment of hypercholesterolemia to prevent cardiovascular diseases, such as myocardial infarction, stroke, and sudden cardiac death (Maron et al., 2000; Takemoto and Liao, 2001). Recent evidence indicates that statins also benefit the cardiovascular system via cholesterol-independent effects, including the inhibition of small GTP-binding protein Rho (Liao, 2002). ROCKs were found to be one of the first downstream targets of RhoA (Leung et al., 1995; Matsui et al., 1996; Ishizaki et al., 1996). In endothelial cells, statin-induced inhibition of RhoA geranylgeranylation decreases membrane GTP-bound active RhoA and subsequent ROCK activity, leading to the up-regulation of eNOS (Laufs and Liao, 1998). Therefore, we hypothesized that the same intracellular signaling pathway might also mediate the SIM-induced up-regulation of nNOS in neurons and observed the effects of exogenous isoprenoid intermediates on the SIM-induced overexpression of nNOS mRNA in CATH.a neurons. As was shown in Fig. 6, the up-regulation of nNOS mRNA expression by SIM treatment was completely abolished by the L-mevalonate, FPP, and GGPP. These results suggest that the up-regulation of nNOS by SIM appears to be specific to the inhibition of HMG-CoA reductase, because the addition of mevalonate and its downstream products completely abolish the stimulatory effect of SIM on nNOS expression. Recently, Nakata et al. (2007) demonstrated that, in cultured rat aortic smooth muscle cells, treatment with atorvastatin significantly increased nNOS mRNA and protein expression through the activation of the Akt/nuclear factor-
B pathway. It is not known whether this pathway also mediated the SIM-induced upregulation of nNOS mRNA expression observed in the current experiment. However, the time course of atorvastatin-induced nNOS expression in smooth muscle cells is almost exactly the same as that of SIM-induced nNOS expression in CATH.a cells, with the initial increase in nNOS expression at day 1 and the peak effect at day 2 implying a potential common intracellular signaling pathway mediating the statin-induced up-regulation of nNOS in smooth muscle cells and neurons.
In conclusion, we demonstrated that centrally administered SIM decreased RSNA and improved arterial baroreflex function via up-regulation of nNOS expression in the RVLM of CHF rabbits. We further documented that the inhibition of HMG-CoA reductase and its downstream pathway mediated the up-regulation of nNOS by SIM, in which the RhoA/ROCK pathway plays a role.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CHF, chronic heart failure; SIM, simvastatin; FPP, farnesyl-pyrophosphate; RVLM, rostral ventrolateral medulla; RT-PCR, reverse transcription-polymerase chain reaction; GGPP, geranylgeranyl pyrophosphate; NOS, nitric-oxide synthase; eNOS, endothelial NOS; nNOS, neuronal NOS; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; MAP, mean arterial pressure; HR, heart rate; CSF, cerebrospinal fluid; RSNA, renal sympathetic nerve activity; L-NAME, N-nitro-L-arginine methyl ester; ROCK, Rho-associated kinase; AP, arterial pressure.
Address correspondence to: Dr. Lie Gao, Department of Cellular and Integrative Physiology. University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850. E-mail: lgao{at}unmc.edu
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