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. 2008 Mar 27;586(Pt 10):2581–2591. doi: 10.1113/jphysiol.2008.150730

20-HETE increases renal sympathetic nerve activity via activation of chemically and mechanically sensitive muscle afferents

Zhaohui Gao 1, Satoshi Koba 1, Lawrence Sinoway 1, Jianhua Li 1
PMCID: PMC2464334  PMID: 18372304

Abstract

Arachidonic acid and its metabolites produced via cyclooxygenase (COX) and lipoxygenase pathways have been reported to contribute to the cardiovascular reflexes evoked by stimulating thin fibre muscle afferents during muscle contraction. 20-Hydroxyeicosatetraenoic acid (20-HETE), a primarily metabolized product of arachidonic acid by cytochrome P450 enzymes, can be accumulated in contracting muscles. Thus, the purpose of this study was to determine the role of 20-HETE in modulating the reflex sympathetic responses to activation of chemically and mechanically sensitive muscle afferents. The renal sympathetic nerve activity (RSNA) and cardiovascular responses were examined after injections of 20-HETE into the arterial blood supply of the hindlimb muscles of decerebrated rats. This induced a dose-dependent increases in RSNA and mean arterial pressure (MAP). We also tested the hypothesis that 20-HETE would sensitize muscle afferents and, thereby, augment the RSNA and blood pressure response to muscle stretch. The results show that arterial infusion of 20-HETE significantly enhanced the RSNA and MAP responses to muscle stretch. In contrast, N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine, a potent inhibitor of 20-HETE production, attenuated the reflex muscle responses. Furthermore, the sensitizing effect of 20-HETE on the muscle reflex was significantly attenuated after blocking COX activity with indomethacin. Our data suggest that 20-HETE plays a role in modulating muscle afferent-mediated sympathetic responses, probably through engagement of a COX-dependent mechanism.

The muscle pressor reflex is one of the neural mechanisms evoking the cardiovascular and sympathetic nervous responses to exercise (Coote et al. 1971; McCloskey & Mitchell, 1972; Mitchell et al. 1977). The afferent arm of this reflex is known to be composed of thinly myelinated groups III and unmyelinated group IV muscle afferents (Kaufman & Forster, 1996), which respond to mechanical deformation of the muscle afferent receptive fields (Kniffki et al. 1978; Kaufman et al. 1983) as well as to metabolic stimulation (Kaufman et al. 1984; Rotto & Kaufman, 1988; Sinoway et al. 1993, 1994). Group III afferents are more mechanically sensitive than group IV afferents (Kaufman et al. 1983), but both are stimulated or sensitized by muscle metabolic by-produces such as arachidonic acid (AA), prostaglandins and ATP produced in contracting skeletal muscle (Rotto & Kaufman, 1988; Rotto et al. 1990; Sinoway et al. 1993, 1994; Hanna & Kaufman, 2004; Kindig et al. 2006).

So far, there are three pathways of AA metabolism discovered in most animal tissues – cyclooxygenase (COX), lipoxygenases (LOX), and cytochrome P450s (P450) (Capdevila et al. 1981; Roman, 2002). Previous studies have shown that prostaglandin E2 (PGE2), a product of the AA and COX pathway, accumulates in skeletal muscle during contraction (Herbaczynska-Cedro et al. 1976; Rotto et al. 1989). Group IV muscle afferents are stimulated by AA injection into their arterial supply, and an effect can greatly be attenuated by COX blockade (Rotto & Kaufman, 1988). PGE2 has also been shown to potentiate the excitatory action of bradykinin on the discharge of group IV muscle afferents (Mense, 1981). In addition, COX, as well as LOX products of AA metabolism, has been found to increase the sensitivities of cutaneous thin fibre mechanoreceptors to distortion of their receptive field (Pateromichelakis & Rood, 1982; Martin et al. 1987), and both COX and LOX products sensitize group III muscle afferent to static contraction (Rotto et al. 1990). COX blockade, which prevents prostaglandin and thromboxane production, has been reported to decrease the reflex cardiovascular responses to static muscular contraction (Stebbins et al. 1986). However, to date there has been no attempt to determine the role of metabolites produced via the AA–P450 pathway in regulating sympathetic nervous and cardiovascular responses to active muscle.

A prior work has indicated that AA is primarily metabolized by P450s to 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (Miyata & Roman, 2005). Among these eicosanoids, 20-HETE is formed in a tissue- and cell-specific fashion and plays an important role in the modulation of cardiovascular and renal function (Gebremedhin et al. 2000; Miyata & Roman, 2005). Along with AA and its other pathway metabolites (Herbaczynska-Cedro et al. 1976; Viinikka et al. 1984; Rotto et al. 1989; Victor et al. 1989), 20-HETE has been reported to accumulate in contracting skeletal muscle (Amaral et al. 2003). A study has further shown that 20-HETE can be converted to 20-OH-PGE2 by the COX-dependent mechanism, and increases AA release (Fang et al. 2006). 20-OH-PGE2 plays a similar role in regulating vascular tone as PGE2 (Oyekan, 2005; Fang et al. 2006).

On the basis of these data, in this report we examined role of 20-HETE in modulating the reflex sympathetic responses to activation of chemically and mechanically sensitive muscle afferents. We hypothesized that the arterial administration of 20-HETE into the blood supply of the triceps surae muscle would raise blood pressure and sympathetic nervous activity, and that 20-HETE would also sensitize thin-fibre muscle afferents. This, in turn, would lead to a greater pressor response for a given degree of deformation of the muscle afferent receptive field. In addition, we further examined if a COX-dependent mechanism was responsible for the effect of 20-HETE on the sympathetic nerve responses to passive muscle stretch.

Methods

All procedures outlined in this study were performed in compliance with the rules and regulations described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. These procedures were approved by the Animal Care Committee of Pennsylvania State University College of Medicine. Male Sprague–Dawley rats (body weight: 400–600 g) were housed in standard rat cages and regulated on a 12: 12 h light–dark schedule, with food and water available ad libitum. At the end of each experiment, animals were humanely killed by intravenous injection of sodium pentobarbital (120 mg kg−1).

Animal surgical preparation

The rats were anaesthetized by inhalation of an isoflurane–O2 mixture (2–5% isoflurane in 100% O2). An endotracheal tube was inserted and attached to a ventilator (model AWS, Hallowell EMC, Pittsfield, MA, USA). Polyethylene (PE-50) catheters were inserted into an external jugular vein and the carotid arteries for the purposes of drug administration and measurement of arterial blood pressure, respectively. A continuous infusion of physiological saline (0.1 ml h−1) into the jugular vein was established by using a syringe pump (Medical Industries, Subiaco, West Australia). This maintained fluid balance and basal blood pressure. The femoral artery was carefully isolated in one hindlimb. An incision was made in the artery. A PE-10 catheter was inserted into the femoral artery so that drugs could be injected into the arterial blood supply of the hindlimb muscles of the leg, as previous described (Li et al. 2004a; Li et al. 2004b). The skin covering the triceps surae muscle and femoral region was surgically separated from the muscle below to eliminate inputs from cutaneous afferents in the hindlimb.

The animals were artificially ventilated, and tidal CO2 was monitored by a respiratory gas monitor (Model 5250, Datex-Ohmeda, Madison, WI, USA) and maintained within normal ranges, as previously described (Li et al. 2004a; Li et al. 2004b). Body temperature was carefully maintained at 37.5–38.5°C by a heating pad and external heating lamps.

Arterial blood pressure was measured by connecting the carotid arterial catheter to a pressure transducer (model 12C, Grass Instruments, West Warwick, RI, USA). Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 s. Heart rate (HR) was determined from the arterial pressure pulse. All measured variables were continuously recorded on an eight-channel chart recorder (model TA 4000, Gould, Valley View, OH, USA) and stored on an iMac computer that used the PowerLab system (ADInstruments, Castle Hill, Australia).

The renal sympathetic nerve activity (RSNA) was recorded as previously described (Miki et al. 2002; Koba et al. 2006). Briefly, a bundle of the renal nerves were carefully dissected from other connective tissues. A piece of laboratory film was placed under the isolated nerves, and two tips of a bipolar electrode to record neural activity were placed between the nerves and the film. These were embedded in a silicone gel. Once the gel was hardened, the silicone rubber was fixed to the surrounding tissue with a glue containing α-cyanoacrylate. The RSNA signal was amplified with an amplifier (P511, Grass Instruments, West Warwick, RI, USA) with a band-pass filter of 300 Hz in low-cut frequency and of 3 kHz in high-cut frequency and made audible.

Decerebration was performed as previously described (Smith et al. 2001; Li et al. 2003, 2004a). A transverse section was made anterior to the superior colliculus and extending ventrally to the mamillary bodies. The brain rostral to the section was then removed. This approach afforded the opportunity to examine the effect of arterial injection of 20-HETE on the RSNA and blood pressure without considering the confounding effects of anaesthesia. Once the decerebration was complete, anaesthetic was removed from the inhaled mixture. A recovery period of 60 min after decerebration was employed to allow sufficient time for elimination of the effects of the anaesthesic gas from the preparation.

Experimental protocols

Study series 1: arterial injection of 20-HETE to induce increase of sympathetic nerve activity

The purpose of this protocol was to determine whether 20-HETE activated the reflex muscle responses. 20-HETE, in the concentrations of 3, 30, and 300 nm (0.1–0.15 ml, dissolved in saline; Sigma-Aldrich) was injected into the blood supplies of the triceps surae muscle. The concentrations of 20-HETE were chosen on the basis of previous studies (Miyata & Roman, 2005; Fang et al. 2006). The duration of injections was 1 min. At least 20 min was allowed between the injections.

Study series 2: effect of 20-HETE on sympathetic response evoked by muscle stretch

It has been reported that both COX and LOX products of AA metabolism enhance discharge rate of the group III muscle afferents responding to static contraction (Rotto et al. 1990). In this series of experiments therefore we examined if 20-HETE can sensitize mechanically sensitive afferents.

Muscle stretch (0.5 kg tension) was produced manually over ∼5 s by using a rack and pinion attached to the Achilles' tendon of the decerebrated rats. Each bout of muscle stretch was maintained for 30 s after 0.5 kg of tension was achieved. Muscle stretch was performed 5 min after arterial injection of saline, 3, 30, and 300 nm of 20-HETE. The injected volume was 0.1–0.15 ml and the duration of injections was 1 min. There was a 30 min rest period between bouts of muscle stretch.

Study series 3: effect of N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine (HET0016) on sympathetic response evoked by muscle stretch

HET0016 is a selective inhibitor of 20-HETE synthesizing enzyme (Miyata et al. 2001; Kehl et al. 2002). A previous study has shown that HET0016 can completely block the increase of 20-HETE formation induced by electrical stimulation of skeletal muscle (Amaral et al. 2003). The purpose of this protocol was to examine the RSNA and cardiovascular responses evoked by passive muscle stretch after inhibiting 20-HETE. Muscle stretch with 0.5 kg of muscle tension was performed and maintained for 30 s ∼10 min after the femoral artery injection of 1 μm of HET0016. The reflex sympathetic nerve, blood pressure and HR responses were then observed.

Study series 4: effect of indomethacin (Indo) on 20-HETE sensitizing sympathetic response

Indo can inhibit the activity of the COX enzyme that converts AA to prostaglandins and thromboxanes. A prior study has also shown that 20-HETE is converted to 20-OH-PGE2 by the COX-dependent mechanism (Fang et al. 2006). In this separate experiment, we determined whether the 20-HETE sensitizing effect was mediated by the COX pathway. Thus Indo (0.2 ml; 0.5 mg kg−1) was administered 40 min before arterial injection of 300 nm 20-HETE. The muscle stretch was stretched 5 min later.

Experimental data analysis

Signals of the RSNA were transformed into absolute values, integrated over every 1 s, and the 1 s integrated background noise was subtracted. The absolute values of the RSNA varied between rats. In order to quantify the sympathetic responses to experimental interventions, basal values were obtained by taking mean values for 30 s immediately before each intervention and by evaluating the mean as 100%, and then evaluating the relative changes from baseline during and after intervention. The tension–time index (TTI) for tendon stretch was calculated by integrating the area between the tension trace and its baseline level (Perez-Gonzalez, 1981). Control values were determined by analysing ≥30 s of the data immediately before femoral arterial injection or muscle stretch. The peak response of each variable was determined by the peak change from the control value.

Measured variables were analysed by using a one-way repeated-measure analysis of variance. As appropriate, Tukey's post hoc test was utilized. Values are means ± s.e.m. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS for Windows version 15.0 (SPSS, Chicago, IL, USA).

Results

20-HETE Increased RSNA and arterial blood pressure (n = 8)

Baseline values for MAP and HR before arterial injections of saline and 20-HETE are presented in Table 1. There were no significant differences in baseline MAP and HR before all injections. Figure 1A shows typical recordings of arterial pressure, HR and RSNA after arterial injections of saline and 300 nm of 20-HETE. 20-HETE evoked a dose-dependent increases in RSNA and pressor response (Fig. 1B). Of note is that 20-HETE, in the concentration of 300 nm, had a significant effect (P < 0.05 versus control, other dosages and recovery). The HR response to 20-HETE injections was not significantly altered (Fig. 1B).

Table 1.

Basal and reflexive MAP and HR responses after arterial injection of 20-HETE

MAP (mmHg) HR (beats min−1)
Baseline Peak Baseline Peak
Saline 93 ± 5 95 ± 5 407 ± 8 410 ± 7
3 nm 90 ± 5 94 ± 5 406 ± 7 409 ± 8
30 nm 93 ± 5 99 ± 5 404 ± 7 409 ± 7
300 nm 92 ± 4 102 ± 5* 406 ± 5 411 ± 5
Recovery 91 ± 6 93 ± 6 413 ± 12 417 ± 10
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Values are means ± s.e.m. The number of animals = 8. MAP, mean arterial pressure; HR, heart rate; 20-HETE, 20-hydroxyeicosatetraenoic acid. There are no significant differences among basal values.

*

P < 0.05, versus baseline.

Figure 1. Effects of arterial injection of 20-HETE on the RSNA and pressor responses.

Figure 1

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A, typical recordings of the changes in RSNA, the relative changes in the RSNA, arterial pressure (AP), and HR in control, after arterial administration of 300 nm 20-HETE, and recovery. B, average data. 20-HETE, in the concentration of 300 nm, induced significant increases in the RSNA and MAP (P < 0.05 versus control, other dosages and recovery. Number of animals = 8). Values are means ± s.e.m. RSNA, renal sympathetic nerve activity; MAP, mean arterial pressure; HR, heart rate. All other abbreviations are as defined in text. Arrows indicate start of injections.

20-HETE enhanced RNSA and pressor responses to muscle stretch (n = 10)

Baseline values for MAP and HR before arterial injections of saline and 20-HETE are presented in Table 2. There were no significant differences in basal MAP and HR before injections. In control, muscle stretch increased MAP by 12 ± 1 mmHg and RSNA by 36 ± 3%. 20-HETE at 300 nm significantly increased the RSNA and pressor responses to muscle stretch (Fig. 2). The peak pressor and RNSA responses were 18 ± 2 mmHg and 83 ± 10% (P < 0.05 versus saline control). There was no significant difference in the HR response to muscle stretch after the saline and 20-HETE injections (Fig. 2). Figure 2 further shows that no significant differences in TTI were seen among the interventions.

Table 2.

Basal and reflexive MAP and HR responses to stretch after arterial injection of 20-HETE

MAP (mmHg) HR (beats min−1)
Baseline Peak Baseline Peak
Saline 88 ± 5 100 ± 5* 388 ± 15 391 ± 15
3 nm 96 ± 9 109 ± 9* 387 ± 10 391 ± 9
30 nm 87 ± 3 101 ± 4* 394 ± 10 398 ± 9
300 nm 88 ± 4 106 ± 4* 394 ± 15 399 ± 14
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Values are means ± s.e.m. The number of animals = 10. MAP, mean arterial pressure; HR, heart rate; 20-HETE, 20-hydroxyeicosatetraenoic acid. There are no significant differences among basal values.

*

P < 0.05, versus baseline.

Figure 2. Effects of arterial administrations of 20-HETE on stretch-induced reflex RSNA and pressor responses.

Figure 2

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20-HETE at 300 nm significantly augmented stretch-induced RSNA and MAP responses (*P < 0.05 versus control and 3 nm; number of animals = 10). There was no significant difference in HR response to muscle stretch, and time–tension index (TTI) among the interventions.

HET0016 attenuated RNSA and pressor responses to muscle stretch (n = 9)

Basal MAP and HR obtained before arterial injections of HET0016 are shown in Table 3. HET0016, at the concentration of 1 μm, significantly attenuated the increases in RSNA and MAP elicited by muscle stretch, compared with control and recovery (Fig. 3). There were no significant differences in developed tension among the interventions (Fig. 3). Original recordings of arterial pressure, HR and RSNA in Fig. 4 further show the inhibitory effects of HET0016 on the reflex muscle responses.

Table 3.

Basal and reflexive MAP and HR responses to stretch after arterial injection of HET0016

MAP (mmHg) HR (beats min−1)
Baseline Peak Baseline Peak
Control 100 ± 5 115 ± 8 419 ± 24 422 ± 25
0.01 μm 91 ± 2 101 ± 4* 422 ± 25 426 ± 25
0.1 μm 94 ± 7 103 ± 8 400 ± 19 405 ± 18
1 μm 85 ± 3 90 ± 3 409 ± 9 414 ± 10
Recovery 95 ± 10 110 ± 11* 398 ± 38 403 ± 38
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Values are means ± s.e.m. The number of animals = 9. MAP, mean arterial pressure; HR, heart rate; HET0016, N-hydroxy-N′-(4-butyl-2-methylphenyl) formamidine. There are no significant differences among basal values.

*

P < 0.05, versus baseline.

Figure 3. Effects of arterial injection of HET0016 on stretch-induced reflex RSNA and pressor responses.

Figure 3

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HET0016, at the concentration of 1 μm, significantly attenuated the increases in RSNA and MAP elicited by muscle stretch (*P < 0.05 versus control and recovery; number of animals = 9). There were no significant differences in developed tension among the interventions.

Figure 4. Typical recordings showing the changes in the RSNA, arterial pressure, HR and muscle tension in control, after arterial administrations of HET0016 and recovery.

Figure 4

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HET0016 attenuated the increases in RSNA and MAP elicited by muscle stretch.

Indo blunted 20-HETE sensitizing muscle reflex (n = 9)

Basal cardiovascular values obtained before drug injections are shown in Table 4. Muscle stretch increased MAP by 12 ± 2 mmHg, and RSNA by 32 ± 4% after saline injection.20-HETE at 300 nm significantly enhanced the reflex responses (Fig. 5). The MAP and RSNA responses to muscle stretch were 18 ± 2 mmHg and 69 ± 5% after 20-HETE. Furthermore, enhanced MAP and RSNA responses by 20-HETE were attenuated by the prior administration of Indo, 0.5 mg kg−1 (Fig. 5). It is noted that blocking COX with this concentration of Indo did not significantly alter baseline MAP and HR and their responses. Typical recordings of the changes in MAP, HR, and RSNA are shown in Fig. 6.

Table 4.

Basal and reflexive MAP and HR responses to stretch after HET0016 with prior injection of Indo

MAP (mmHg) HR (beats min−1)
Protocol Baseline Peak Baseline Peak
Control 102 ± 4 114 ± 4* 387 ± 17 391 ± 19
20-HETE 94 ± 3 112 ± 2* 388 ± 13 393 ± 12
Indo 99 ± 4 109 ± 4 392 ± 19 396 ± 20
Indo + 94 ± 3 106 ± 4* 417 ± 18 420 ± 18
20-HETE
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Values are means ± s.e.m. The number of animals = 9. MAP, mean arterial pressure; HR, heart rate; 20-HETE, 20-hydroxyeicosatetraenoic acid; Indo, indomethacin. There are no significant differences among basal values.

*

P < 0.05, versus baseline.

Figure 5. Effects of arterial administration of Indo on 20-HETE-sensitizing muscle reflex.

Figure 5

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20-HETE at 300 nm significantly enhanced the reflex responses (*P < 0.05 versus other injections; number of animals = 9). The augmented RSNA and MAP responses by 20-HETE were attenuated by the prior administration of Indo (0.5 mg kg−1). There were no significant differences in developed tension among the interventions.

Figure 6. Typical recordings showing that 20-HETE increased RSNA and blood pressure responses to muscle stretch, and that the enhanced muscle reflex was attenuated by the prior administration of Indo.

Figure 6

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All abbreviations are as defined in text.

Discussion

Study findings

The current studies were performed to determine whether 20-HETE, a P450 hydroxylases metabolite of AA, is involved in the sympathetic nervous and cardiovascular responses in active muscle. The major findings of this study are that (1) arterial infusions of 20-HETE evoked dose-dependent increases in RSNA and arterial blood pressure; (2) 20-HETE also sensitized sympathetic nervous activity and the pressor response evoked by mechanically sensitive skeletal muscle stretch; (3) arterial infusions of HET0016, an inhibitor of 20-HETE formation, attenuated RSNA and pressor response to muscle stretch; (4) the enhanced effect of 20-HETE on the reflex muscle responses to stretch was attenuated by Indo, a COX blocker. These findings suggest that 20-HETE plays a role both in stimulating chemically sensitive afferents and in sensitizing mechanically sensitive afferents, and its effects are likely to be mediated by a COX-dependent mechanism.

It has been known that PGE2 and AA contribute to the reflex cardiovascular responses to muscle contraction (Stebbins et al. 1986; Kaufman & Rybicki, 1987; Rotto & Kaufman, 1988; Rotto et al. 1989; Hayes et al. 2006). Our present data have further shown that a P450 metabolite, 20-HETE, increased the RSNA and blood pressure when it was injected into the arterial blood supply of hindlimb muscle. In addition, in this study, HET0016, an inhibitor of P450, was administrated into the femoral artery. It is very interesting that our data have shown that 1 μm of HET0016 significantly attenuated the sympathetic and cardiovascular responses to muscle stretch. Thus it is reasonable to consider that 20-HETE on its own may play an important role in stimulating muscle afferents.

Previous studies have shown that COX plays an important role in 20-HETE metabolisms and its physiological activity. First, 20-HETE is converted to 20-OH-PGE2 (one of two major metabolites from 20-HETE) by the COX-dependent mechanism, and 20-OH-PGE2 has the same biological effects as PGE2 (Fang et al. 2006). Furthermore, the addition of 20-HETE to mouse brain endothelial cultures was found to increase the production of PGE2 and prostacyclin (Fang et al. 2006). The mechanism by which PGE2 and prostacyclin are increased is likely to be related to increases in AA, perhaps triggered by a 20-HETE-mediated increase in intracellular free Ca2+ (Muthalif et al. 1998). Also, it should be noted that previous studies demonstrating constrictor effects of 20-HETE in the cerebral artery were performed in the presence of Indo, which masked a vasodilatory effect of 20-HETE that was dependent on COX activity (Alonso-Galicia et al. 1999; Yu et al. 2004). Finally, another 20-HETE metabolite, 20-COOH-AA, can increase formation of PGE2. All these findings indicated that 20-HETE could directly or indirectly increases production of PGE2 and AA.

In this study, the enhanced effect of 20-HETE on the reflex muscle responses to stretch was attenuated after COX blockade. This suggests that 20-HETE is likely to exert its effects by increasing 20-OH-PGE2 via a COX-dependent mechanism. Thus we would speculate that 20-OH-PGE2 is an important stimulator/sensitizer of muscle afferents.

Previous studies have reported that Indo (given at 5 mg kg−1, i.v.) attenuates the discharge of muscle afferents, and the reflex pressor response to active muscle (Rotto et al. 1990; Hayes et al. 2006). In order to minimize the masking effect of Indo on 20-HETE induced augmentation of the muscle reflex, we injected a lower dose of Indo (0.5 mg kg−1, i.a.) in this study. Our result shows that Indo did not alter baseline MAP and HR, and their responses. We believe that this dose of Indo was not sufficient to attenuate the muscle reflex. However, enhanced MAP and RSNA responses by 20-HETE were attenuated by the prior administration Indo. This further suggests that blocking the COX pathway with Indo attenuates the effects of 20-HETE.

A recent study has shown that 20-HETE contributes to purinergic P2X receptor-mediated afferent arteriolar vasoconstriction (Zhao et al. 2001). 20-HETE can also activate vanilloid type 1 receptor (TRPV1) on C-fibre nerve endings and result in depolarization of nerves and consequent vasoactive neuropeptide release (Scotland et al. 2004). Although there are no direct data to show an interaction between 20-HETE and acid-sensing ion channels (ASICs), it was found that AA, the precursor of 20-HETE, might enhance ASIC activity (Allen & Attwell, 2002). P2X receptors, TRPV1 and ASICs appear on thinly unmyelinated and myelinated afferent nerve fibres (Cook et al. 1997; Chen et al. 1998; Ma, 2001). It has previously been reported that these receptors play a role in processing of muscle afferent signals in evoking and modulating the muscle pressor reflex in decerebrated cats and rats (Li & Sinoway, 2002; Li et al. 2004a; Gao et al. 2006; Hanna & Kaufman, 2004; Kindig et al. 2006; Hayes et al. 2007). Taken together with the data from the current report, it is suggested that 20-HETE may act to form 20-OH-PGE2 and as an intracellular signalling molecular rather than act on a special receptor located in thin-fibre afferents. The precise molecular and cellular mechanism remains to be explored.

Abnormal 20-HETE formations have been reported in a variety of cardiovascular diseases such as hypertension and cerebrovascular disorders (Roman, 2002; Granville & Gottlieb, 2006). Inhibitors of the synthesis of 20-HETE have been shown to have beneficial effects in treatment of those diseases (Miyata et al. 2001). Our study, for the first time, investigated 20-HETE mediated reflexive sympathetic and pressor responses. Thus, this has the potential to better our understanding of the contribution of 20-HETE in pathophysiological conditions such as heart failure, in which muscle mechanoreceptor overactivity mediates the exaggerated sympathetic responses (Li et al. 2004b; Smith et al. 2005; Middlekauff & Sinoway, 2007).

Finally, numerous studies have shown that 20-HETE can cause vasoconstriction in skeletal muscle (Roman, 2002). If 20-HETE constricts the hindlimb vasculature and makes the muscles partially ischaemic, we cannot rule out the possibility that muscle afferent nerves are sensitized, and the sympathetic responses to muscle stretch are enhanced by the decrease in blood flow caused by 20-HETE vasoconstriction.

Conclusion

The administration of 20-HETE into the arterial blood supply of the triceps surae muscle increases blood pressure and sympathetic nervous activity. Also, 20-HETE can sensitize thin-fibre muscle afferents and enhance the sympathetic response to muscle stretch. In addition, we further suggest that the effect of 20-HETE on the sympathetic nerve response is likely to be due to a COX-dependent pathway.

Acknowledgments

The authors express gratitude to Jennie Stoner for outstanding secretarial skills. This study was supported by NIH R01 HL075533 (J.L.), R01 HL078866 (J.L.) and R01 HL060800 (L.S.).

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