Dai and colleagues (Dai X, Galligan JJ, Watts SW, Fink GD, and Kreulen DL. Hypertension 43: 1048–1054, 2004) found that endothelin (ET) stimulated O2− production in sympathetic ganglion neurons in vitro by activating ETB receptors. The objective of the present study was to determine whether activation of ETB receptors in vivo elevates O2− levels in sympathetic ganglia. Because ETB receptor activation increases blood pressure, we also sought to determine whether alteration in O2− levels was a direct effect of ETB receptor activation on sympathetic ganglia or an indirect consequence of hypertension. Male Sprague-Dawley rats received intravenous infusions of either the specific ETB receptor agonist sarafotoxin 6c (S6c; 5 pmol·kg−1·min−1) or isotonic saline at 0.01 ml/min (control) for 120 min. To measure O2− levels, we removed the inferior mesenteric ganglion immediately after infusion and stained it with dihydroethidine (DHE). Mean arterial pressure increased 26.6 ± 1.7 mmHg in the S6c-treated rats and 3.65 ± 6 mmHg in control rats. Measurements of average pixel intensity revealed that the DHE fluorescence in ganglionic neurons and surrounding glial cells were 96.7% and 160% greater in S6c-treated than in control rats, respectively. To evaluate the effect of elevated blood pressure on O2− production, a separate group of rats received phenylephrine (PE; 10 μg·kg−1·min−1 iv) for 2 h. MAP increased 31 ± 1.2 mmHg in PE-infused rats. The DHE fluorescence intensity in ganglia of PE-infused rats was significantly greater than that of control rats, 137.7% in neurons and 104.6% in glia but significantly lower than in ganglia from S6c rats. We conclude that ETB receptor activation in vivo significantly enhances O2− levels in sympathetic ganglia, due to both pressor effects and direct stimulation of ETB receptors in ganglion cells.
- sarafotoxin 6c
- sympathetic nervous activity
- oxidative stress
- reactive oxygen species
hypertension caused by numerous genetic and neurohumoral factors is associated with higher amounts of reactive oxygen species (ROS) in blood vessels, brain, and kidneys; examples include ANG II-mediated hypertension, deoxycorticosterone acetate (DOCA)-salt hypertension, mineralocorticoid hypertension, aortic banding-induced hypertension, renovascular hypertension, and endothelin-induced hypertension (1, 3, 18–20, 23, 28, 36, 42). The best characterized ROS in tissues of hypertensive individuals is superoxide anion (O2−). Reduction in O2− formation can lower blood pressure in some experimental models of hypertension (1, 9, 14, 32, 38), suggesting that increased production of ROS is an etiologic factor in hypertension. O2− can increase blood pressure by several mechanisms. In the vasculature, O2− causes vasoconstriction, in part by inducing endothelial cell dysfunction (5). Increased O2− in the kidney is associated with enhanced tubular reabsorption of sodium and water (27, 33). In key brain regions, increased O2− leads to increased sympathetic nervous system activity (SNA) (7, 51–53). The focus of the work to be reported here, however, is on the peripheral sympathetic nervous system. We previously presented evidence that O2− enhances peripheral sympathetic neurotransmission and that this action is accentuated in rats with DOCA-salt hypertension (47, 48).
The DOCA-salt model of experimental hypertension depends in part on the activity of the endothelin (ET) system (24). Considerable evidence indicates that ET can both stimulate the formation of O2− (26, 27) and increase SNA (2, 12, 16, 43). Furthermore, several studies have found that antioxidants (14, 32, 38, 49) or a reduction in SNA (30, 31) can attenuate the hypertensive effects of ET. A critical finding for the present study was the observation by Dai et al. (10) that sympathetic neurons in peripheral ganglia contain O2− and that the content of O2− is significantly increased in ganglia from DOCA-salt rats. They went on to test for a possible influence of ET on O2− production by sympathetic ganglia. Although ET generally increases O2− levels by stimulating the ETA receptor subtype (6), Dai et al. (10) showed that ET increases O2− levels in sympathetic ganglia by activating ETB receptors. They also reported that the expression of ETB receptors is higher in ganglia from hypertensive DOCA-salt rats than in normotensive control rats. Their findings suggest that ET may increase SNA in DOCA-salt hypertension through an action at ETB receptors located on cell bodies of postganglionic sympathetic neurons.
We and others have shown that infusion of the selective ETB receptor agonist sarafotoxin 6c (S6c) into conscious rats results in an increase in blood pressure (25, 29). Though ETB receptors are known to cause transient hypotension by the release of the vasodilatory peptides nitric oxide, and prostacyclin (17), they also function as clearance receptors to remove circulating ET-1 (15). Blockade of ETB receptors increases blood pressure presumably by decreasing bioavailability of ET-1 in the circulation, thus potentiating activation of ETA receptors (22, 34, 37). The present study was designed to determine whether activation of ETB receptors in vivo increases O2− levels in sympathetic ganglia. To this end, we infused S6c into rats and measured the amount of O2− production in sympathetic ganglia using the dihydroethidium oxidative fluorescence method. To test whether any changes in O2− levels in ganglia are a consequence of hypertension or of direct ETB receptor activation in ganglia, we also examined superoxide production in response to elevated blood pressure induced by the α adrenergic agonist phenylephrine (PE).
Adult, male Sprague-Dawley rats (200–350 g; Charles River Laboratories, Portage, ME) were assigned to either of two experimental protocols: in vivo or in vitro. All animals were fed standard rat chow and had ad libitum access to both food and water. Animal procedures were approved by the Michigan State University All University Council on Animal Use and Care.
In vivo studies.
In rats under pentobarbital sodium (50 mg/kg ip) anesthesia, catheters were positioned in the abdominal aorta via the left femoral artery for continuous hemodynamic monitoring and in the femoral vein for drug administration. Rats were then housed in standard stainless steel metabolic cages for the duration of the study. Free ends of the catheters exited the cage through a stainless steel tether connected to the rat by a plastic harness around the thorax. After 2–3 days of surgical recovery, rats were subjected to one of three different treatments: they received intravenous infusions of either 1) the specific ETB receptor agonist S6c (5 pmol·kg−1·min−1; American Peptide, Sunnydale, CA), 2) isotonic saline at 0.01 ml/min (control), or 3) the alpha-adrenoceptor agonist PE (10 μg·kg−1·min−1; Sigma-Aldrich, St. Louis, MO) for 2 h. Blood pressure measurements were obtained continuously throughout the protocol without disturbing the animal. Immediately after systemic infusion, animals were euthanized with pentobarbital sodium (100 mg/kg iv), and the inferior mesenteric ganglion (IMG) was excised for superoxide measurement.
In vitro studies.
Animals were killed with a lethal dose of pentobarbital sodium (100 mg/kg ip), and their IMG was immediately harvested. To evaluate whether in vitro administration of agonist to the ganglia might affect levels of O2−, isolated IMG were incubated with varying concentrations of PE (1 μM to 100 μM) for 30 min at 37°C. Another set of IMG were treated with S6c (10−8 mol/l), as described by Dai et al. (10) for 30 min at 37°C to serve as positive control, while negative control IMG received no treatment.
Ganglionic O2− production was assessed by oxidative dihydroethidium fluorescence method, as previously described (10). In brief, IMG were incubated with the oxidant-sensitive probe DHE (2 μmol/l; Molecular Probes) for 45 min at 37°C. The levels of O2− were assayed by measuring the fluorescence signal intensity resulting from intracellular oxidation of the DHE to fluorescent ethidium by O2−. The fluorescent intensity is proportional to O2− levels. The fluorescent signal (excitation: 514 nm; emission: 560 nm) was measured with a confocal microscope and analyzed using ImageJ Software (U.S. National Institutes of Health, Bethesda, MD). Because DHE fluorescence measurements only provide semiquantitative information, the assay was performed on control groups of animals alongside the treatment groups for every experiment using the same parameters, that is, animals were killed and tissue harvested at (approximately) the same time, on the same day, using the same reagents, on the same microscope and software; thus, providing a baseline standard for each comparison. Larger cells (20–35 μm) were identified as neurons, whereas smaller cells in the periphery (5–10 μm) were identified as glia.
All data were expressed as means ± SE. Statistical significance was assessed with one-way ANOVA with Tukey's post hoc test using Prism 3.0 Software (GraphPad). Paired t-tests were used to compare blood pressure values before and after treatment. A value of P < 0.05 was considered significant.
Effect of in vivo S6c infusion on MAP and O2− production.
S6c infusion for 2 h in conscious rats significantly increased blood pressure. MAP (the difference between the 2 h value and the initial value) increased 26.6 ± 1.7 mmHg in the S6c-treated rats (n = 6) and 3.6 ± 6.0 mmHg in control rats (n = 5) (Fig. 1). S6c infusion also significantly augmented O2− levels in both neurons and glial cells of the IMG compared with control rats. DHE fluorescence measured by average pixel intensity in the ganglionic neurons and surrounding glial cells was 96.7% and 160% greater in S6c than in control rats, respectively (Fig. 2).
Effect of increased MAP on O2− production.
To determine whether the alteration in O2− levels observed in rats receiving S6c was a direct effect of ETB receptor activation on sympathetic ganglia or an indirect consequence of hypertension, in a separate study, rats received either S6c, PE (at a dose chosen to mimic the pressor response to S6c), or isotonic saline treatments. MAP increased 29.9 ± 0.1 mmHg in S6c, 31 ± 1.2 mmHg in PE, and 1.7 ± 1 mmHg in control rats (Fig. 3). As observed in the previous experiment, in vivo infusion of S6c increased the DHE fluorescence intensities of ganglionic neurons and surrounding glial cells significantly more than in control rats, 215.5% and 197.6%, respectively. Fluorescence intensities of ganglia from PE-infused rats were also significantly greater than controls, 137.7% in neurons and 104.6% in glia, but significantly lower than in ganglia from S6c rats (Fig. 4).
Effects of PE on ganglionic cells.
To determine whether PE acts directly on ganglia to increase O2− levels, we incubated freshly dissociated IMG from normal rats with either PE at 1 μM and at 100 μM or with S6c at 10−8 M. Results (Fig. 5) show that PE has little direct effect in vitro on O2− levels in sympathetic ganglia, whereas S6c produced a large increase.
The main new finding of this study is that activation of ETB receptors in vivo increases O2− levels in sympathetic ganglia. Our observation is consistent with an earlier report that ET peptides stimulate O2− production in sympathetic ganglion neurons in vitro by activating ETB receptors (10). In the current study, we also confirmed that previous finding.
The ability of ETB receptor activation to increase O2− levels in sympathetic ganglia in vivo may be due to both direct and indirect mechanisms. Experiments performed in sympathetic ganglia in vitro show that ET can increase O2− levels by stimulating NAD(P)H oxidase (11). This mechanism also appears to account for the increased O2− levels measured in sympathetic ganglia from DOCA-salt rats (11). In the current study, we did not test whether activation of NAD(P)H oxidase contributes to elevated O2− levels in rats receiving acute infusions of S6c.
Growing evidence points to the possibility that hypertension per se can increase O2− levels in various tissues (13, 45, 46), although other studies indicate hypertension is not invariably associated with increased O2− levels (36). Therefore, to test the hypothesis that S6c increases O2− levels in sympathetic ganglia in part by elevating blood pressure, we infused PE (10 μg·kg−1·min−1) into conscious rats to produce an increase in blood pressure similar to that observed during S6c infusion. Additional rats received either S6c or saline infusions to allow direct comparison of DHE fluorescence with the three stimuli. The results confirmed our previous experiment showing that DHE fluorescence intensities of ganglionic neurons and surrounding glial cells were significantly greater in rats receiving S6c than in control rats. Interestingly, PE infusion also produced O2− levels that were significantly greater than those observed in saline control animals. It is important to note, however, that they remained significantly less than those found in S6c-infused animals. To determine whether PE has any direct effect on superoxide anion levels, we performed an additional study in freshly dissociated rat inferior mesenteric ganglionic neurons and glial cells in vitro. We found that application of PE did not induce a significant increase in superoxide anion levels in either neurons or glial cells. We conclude that an acute increase in blood pressure alone can cause elevated O2− levels in sympathetic ganglia, although it is possible that some other physiological response to PE infusion is responsible. Overall, then these data indicate that S6c infusion in vivo may increase O2− levels in sympathetic ganglia by both direct (stimulation of ETB receptors on neurons and glia) and indirect (pressure-dependent) mechanisms. The indirect mechanism may play a predominant role.
Our findings demonstrate for the first time that in vivo ETB receptor activation increases superoxide anion levels in sympathetic ganglia. Although the actions of superoxide anion in the vasculature, kidney, and brain have been well described, its role in the peripheral sympathetic nervous system is less well characterized. We previously presented evidence that O2− enhances peripheral sympathetic neurotransmission and that this action is accentuated in rats with DOCA-salt hypertension (47, 48). Others have confirmed that finding in spontaneously hypertensive rats (40). They suggested that a potential mechanism of O2− action on sympathetic neurotransmission is by affecting voltage-gated potassium channels in sympathetic nerve fibers (41). Another possibility is that O2− may reduce the bioavailability of nitric oxide in sympathetic ganglia (8). Nitric oxide can alter potassium currents in sympathetic ganglion neurons (4) and the effects of nitric oxide on ganglionic neurotransmission are generally inhibitory (35). Furthermore, it has been shown that activation of ETB receptors in sympathetic ganglia causes an increase in nitric oxide, which acts to inhibit nicotinic transmission through the ganglion (50). Generation of O2− in response to ETB receptor activation might then moderate the inhibitory action of nitric oxide, that is, enhance neurotransmission through the ganglion.
Alternatively, prolonged oxidative stress due to elevated O2− levels in sympathetic ganglia could impair ganglionic transmission by hastening apoptosis of postganglionic sympathetic neurons (21, 44). Currently, however, there is no evidence for decreased neurotransmission through sympathetic ganglia in animals with DOCA-salt or other forms of ET-dependent hypertension.
Elevated superoxide (O2−) anion concentrations in sympathetic ganglia may participate in the pathogenesis of ET-dependent hypertension by facilitating nicotinic neurotransmission through the ganglion. Sympathetic ganglia represent a potential target for antioxidant-based therapy of hypertension and other cardiovascular diseases.
This research was supported by National Institutes of Health Grant P-01-HL-70687.
The authors thank Dr. Xiaoling Dai for her advice and assistance on the DHE method.
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