In women, sympathoexcitation during static handgrip exercise is reduced during the follicular phase of the ovarian cycle compared with the menstrual phase. Previous animal studies have demonstrated that estrogen modulates the exercise pressor reflex, a sympathoexcitatory mechanism originating in contracting skeletal muscle. The present study was conducted in female rats to determine whether skeletal muscle contraction-evoked reflex sympathoexcitation fluctuates with the estrous cycle. The estrous cycle was judged by vaginal smear. Plasma concentrations of estrogen were significantly (P < 0.05) higher in rats during the proestrus phase of the estrus cycle than those during the diestrus phase. In decerebrate rats, either electrically induced 30-s continuous static contraction of the hindlimb muscle or 30-s passive stretch of Achilles tendon (a maneuver that selectively stimulates mechanically sensitive muscle afferents) evoked less renal sympathoexcitatory and pressor responses in the proestrus animals than in the diestrus animals. Renal sympathoexcitatory response to 1-min intermittent (1- to 4-s stimulation to relaxation) bouts of static contraction was also significantly less in the proestrus rats than that in the diestrus rats. In ovariectomized female rats, 17β-estradiol applied into a well covering the dorsal surface of the lumbar spinal cord significantly reduced skeletal muscle contraction-evoked responses. These observations demonstrate that the exercise pressor reflex function and its mechanical component fluctuate with the estrous cycle in rats. Estrogen may cause these fluctuations through its attenuating effects on the spinal component of the reflex arc.
- exercise pressor reflex
- skeletal muscle contraction
- estrous cycle
- renal sympathetic nerve activity
in women, muscle sympathetic nerve response to static handgrip exercise at a given level is reduced during the late follicular phase of the ovarian cycle compared with the menstrual phase (5). The fluctuation of the sympathoexcitatory response to exercise with the ovarian cycle is likely due to the effect of estrogen, which increases to a peak during the late follicular phase. A reflex originating in contracting skeletal muscles contributes to sympathoexcitation seen during exercise (3, 13, 21). This reflex, termed the exercise pressor reflex, originates from chemical and mechanical activation of nerve endings of thin-fiber muscle afferents (i.e., group III and IV). Signals from the nerve endings project to the dorsal horn of the spinal cord and then to the brain stem (11). Previous animal studies have suggested that estrogen modulates the exercise pressor reflex. Schmitt and colleagues (28–31) have reported that topical application of estrogen to the lumbosacral spinal cord reduced the pressor response to continuous static contraction of hindlimb skeletal muscle in cats and rats 60 min after its application, suggesting that, in these animals, spinal estrogen attenuates the exercise pressor reflex. The threshold concentration of estrogen that reduced the exercise pressor reflex in female cats was 1,000 times more dilute than it was in male cats, suggesting that the estrogen effect is sex dependent, more predominant in females (29, 31). Because application to the spinal cord of naloxone, a μ-opioid and δ-opioid antagonist, partially prevented the effect of spinal estrogen to attenuate the pressor response to skeletal muscle contraction in female cats, it was suggested that opioid-dependent mechanisms contribute to estrogen's effect to attenuate the exercise pressor reflex (30).
While roles played by estrogen in attenuating the exercise pressor reflex have been demonstrated, little attention has been paid to the effect of the ovarian cycle on this reflex. In this regard, muscle sympathetic nerve activity elevation seen during posthandgrip exercise ischemia, a maneuver that selectively stimulates chemically sensitive muscle afferents, in female subjects was shown to be reduced during the follicular phase compared with the menstrual phase (5). This result suggests that the ovarian cycle in women affects the chemical component of the exercise pressor reflex. However, the procedure of the postexercise ischemia is not equal to induction of the exercise pressor reflex by skeletal muscle contraction. Moreover, the effect of the ovarian cycle on the mechanical component of the reflex (muscle mechanoreflex) remains unknown. Therefore, the present study was conducted in female rats to determine whether reflex sympathoexcitation in response to skeletal muscle contraction or activation of the muscle mechanoreflex fluctuates with the estrous cycle.
MATERIALS AND METHODS
All procedures outlined in the present study complied with the “Guiding Principles for the Care and Use of Animals in the Fields of Physiological Sciences” published by the Physiological Society of Japan, and were approved by the Animal Care Committee of the Tottori University Faculty of Medicine. Experiments in this study were performed on 61 adult (10–16 wk) female Wistar rats. Rats were housed in standard rodent cages in a temperature-controlled room (24°C) and regulated on a 12:12-h light-dark schedule. Food and water were made available ad libitum.
Vaginal smear to assess the estrous cycle.
The estrous cycle in rats includes proestrus, estrus, metestrus, and diestrus phases. Estrogen concentration is the lowest during the estrus phase, increases gradually during the metestrus and diestrus phases, and peaks during the proestrus phase (7). The stage of the estrous cycle in rats was determined by vaginal smear cytology (20). The vaginal lavage monitoring was preliminarily performed before the experimental day to predict the estrous cycle phases on the experimental day. Immediately before the data collection on the experimental day described below, the vaginal lavage was collected, and the estrous cycle phase would be determined by an investigator who had been blinded to the experimental data. Because in rats the rising level of estradiol peaks around the midday on proestrus (7), we classified the data into two groups: one was collected from rats during the proestrus phase (Pro) and another was collected from rats during the diestrus phases, including the metestrus phase (Di). Although estrogen drops at the early period of the estrus phase, there is a prolonged “genomic” effect of estrogen during the estrus phase from the previous proestrus phase. This possibly influences the results in certain physiological studies, especially when the impact of estrogen is determined by a comparison made between the proestrus (high estrogen) and estrus (low estrogen) phases (12). Therefore, the rats during the estrus phase were not studied in the present study.
Surgical procedures and experiments to observe the exercise pressor reflex responses.
The rats were anesthetized with isoflurane (1–4%) in oxygen. The trachea was cannulated, and the lungs were mechanically ventilated (SN480-7, Shinano). The left jugular vein and common carotid artery were cannulated to administer drugs and to measure arterial pressure, respectively. The arterial catheter was attached to a pressure transducer (P23XL, BD). Heart rate (HR) was calculated beat to beat from the arterial pressure pulse. Arterial pH was monitored with a pH meter (B-212, Horiba) and maintained within normal range by an intravenous administration of a sodium bicarbonate solution (8.4%). Rectal body temperature was monitored with a digital thermometer and maintained between 37.5 and 38.5°C with a heating pad. To measure renal sympathetic nerve activity (RSNA), a bipolar electrode made of a Teflon-insulated stainless steel wire (790600, A-M Systems) was connected to the renal nerve directed to the left kidney, as described in our laboratory's previous experiments (16, 17). The RSNA signal was amplified with an AC amplifier (P511, Grass Instruments) with a band-pass low-frequency filter of 100 Hz and a high-frequency filter of 3 kHz, and made audible. The left Achilles tendon and left triceps surae muscles were isolated by cutting the calcaneus bone and dissecting the tendon and muscles free from the connective tissue that attached to the tibia. The hindlimb was fixed in space with a patellar precision clamp to prevent limb movement. The tension developed within the triceps surae muscles was measured with a force transducer (FT03, Grass Instruments) connected to the Achilles tendon. The rats were held in a stereotaxic head unit with customized spinal frame (900LS, David Kopf Instruments). A laminectomy exposed the lower lumbar portions of the spinal cord (L2–L6). The meningeal layers surrounding the cord were cut and reflected laterally. Two nerve bundles obtained from L4 and L5 ventral roots were carefully isolated and sectioned. The peripheral cut ends of the roots were placed on a bipolar stimulating electrode. The exposed neural tissue was immersed in mineral oil and maintained at a temperature of 37°C. A decerebration was performed at the midcollicular level, as conducted previously (16, 17).
We compared the muscle afferent-mediated sympathoexcitatory and cardiovascular responses between the Pro and Di rats. To stimulate thin-fiber muscle afferents, we employed three maneuvers. The first was 30-s continuous static contraction of the hindlimb skeletal muscle, which excites both chemically and mechanically sensitive muscle afferents (10, 14, 36). The second maneuver was 30-s continuous passive stretch of Achilles tendon, which is considered a potent and specific muscle mechanoreceptor stimulant (10, 34). The third was 1-min intermittent bouts (1- to 4-s stimulation to relaxation, 12 contractions) of static contraction of the hindlimb skeletal muscle, which excites predominately mechanically sensitive muscle afferents (16, 17, 23, 35). Contraction of the left hindlimb muscles was induced by excitation of ventral roots with constant-current electrical stimulation [2 × motor threshold (MT), 0.1 ms, 40 Hz]. The minimum current intensity necessary to induce a muscle twitch served as MT. A recovery period of at least 90 min after the decerebration was allowed before the experimental protocol was begun. The decerebrate rats were mechanically ventilated (tidal volume 5.5–6.0 ml/kg and frequency 70 breaths/min). After 30 s of baseline data were collected, each maneuver to stimulate muscle afferents was conducted, and the RSNA and cardiovascular responses were observed.
Bilateral ovariectomy was performed in another set of female Wistar rats 10–12 wk of age, anesthetized with isoflurane (1–4%) in oxygen. We employed ovariectomized rats to eliminate the effect of cyclic changes in estrogen on autonomic nervous system function. Four to six weeks after the surgery, we examined the effect of spinal estrogen to modulate the exercise pressor reflex by observing RSNA and cardiovascular responses to 30-s continuous static contraction of the hindlimb skeletal muscle before and 60 min after 17β-estradiol topically applied to the spinal cord. To apply 17β-estradiol, the dorsal surface of the L4–L5 segments of the spinal cord were enclosed within a “well” created by applying layers of vinyl polysiloxane. Estrogen receptors-α/β are present in spinal dorsal horn neurons (1, 24). To ensure its integrity, the well was filled with saline and checked for leakage. The well was then filled either with saline or water-soluble 17β-estradiol (E4389, Sigma), dissolved in saline (0.01 μg/ml, 100 μl). The application procedure followed previous ones conducted by Schmitt and colleagues (28–31).
At the end of data collection, neuromuscular transmission was blocked with an intravenous infusion of pancuronium bromide (0.25 mg). Then the ventral roots were stimulated at 2 × MT, to confirm that the observed responses to muscle contraction were not due either to current spread to the spinal cord, or to direct stimulation of group III or IV afferents. In all rats, the stimulation after neuromuscular blockade did not change arterial pressure, HR, or RSNA. After all of the experiments were conducted, the renal nerve was cut between the neural axis and the electrode to measure the background noise of RSNA. At the conclusion of the experiment, the rats were humanely killed with an intravenous infusion of pentobarbital sodium (75 mg/kg), followed by an intravenous infusion of potassium chloride (2 M, 1 ml).
Plasma estrogen concentrations.
Venous blood (0.9 ml) was withdrawn to examine plasma concentrations of estrogen with the enzyme-linked immunosorbent assay. The venous blood was mixed with 0.1 ml of heparinized saline (100 U/ml). The samples were centrifuged at 14,000 rpm for 10 min. The blood plasma was then collected and stored at −80°C until analysis. Plasma estradiol levels were measured using the estradiol EIA kit (582251, Cayman Chemical). The sensitivity was 19 pg/ml.
Data acquisition and statistical analyses.
All measured variables were continuously displayed on a computer monitor and stored on a hard disk through an analog-digital interface (Powerlab/8s, ADInstruments) at a 1-kHz sampling rate. Baseline data were obtained from the averaged values for 30 s immediately before each muscle stimulation maneuver. RSNA and cardiovascular responses to each muscle stimulation were determined as analyzed previously (16, 17). Briefly, peak responses to muscle stimulation and integrated values of the responses during muscle stimulation were examined. To quantify RSNA response to muscle stimulation, relative changes in RSNA from the baseline level considered 100% (ΔRSNA) were evaluated. The integrated ΔRSNA were calculated by integrating the increase in RSNA due to muscle stimulation.
The data are expressed as means ± SE. To assess a significant difference in data between the Pro and Di rats, the data were analyzed with an unpaired Student t-test. To assess a significant effect of 17β-estradiol on the reflex responses in the ovariectomized rats, the data were analyzed with a paired Student t-test. To assess a significant difference in the muscle reflex responses from baseline, the data were analyzed with one-way repeated ANOVA, followed by a post hoc test (Dunnett's method). The level of significance was set at P < 0.05.
Plasma estrogen concentration.
Plasma concentrations of the estradiol during the proestrus phase (46.2 ± 7.0 pg/ml, N = 15) were significantly higher than those during the diestrus phase (30.2 ± 3.0 pg/ml, N = 16).
Continuous contraction-evoked responses.
Baseline signal-to-noise ratio for the RSNA, mean arterial pressure (MAP), and HR before 30-s continuous contraction of the hindlimb skeletal muscle were not different between the Pro and Di rats (Table 1). This muscle stimulation increased RSNA, MAP, and HR, and the sympathoexcitatory and pressor responses were significantly less in the Pro rats than those in the Di rats, despite the fact that triceps surae muscle tension generated by contraction of the muscle was not different (Figs. 1 and 2). HR response was not significantly different between the groups.
Passive stretch-evoked responses.
Baseline signal-to-noise ratio for the RSNA, MAP, and HR before 30-s passive stretch of the left Achilles tendon was not different between the Pro and Di rats (Table 1). This muscle stimulation elevated RSNA and MAP and had little effects on HR (Figs. 1 and 3). Integrated RSNA response and peak pressor responses to passive stretch were significantly less in the Pro rats than those in the Di rats at the equivalent tension development (Figs. 1 and 3). Peak RSNA response was not significantly different between the groups (P = 0.11).
Intermittent contraction-evoked responses.
Baseline signal-to-noise ratio for the RSNA, MAP, and HR before 1-min intermittent bouts of static contraction of the hindlimb skeletal muscle were not different between the Pro and Di rats (Table 1). During 1 min of intermittent contraction in these rats, RSNA responded synchronously as muscle tension was developed (Fig. 4). Intermittent contraction had little effect on the averaged changes in MAP from baseline in each rat group because this contraction protocol induced variable patterns in the MAP changes across the rats. These characteristics of RSNA and arterial pressure dynamics seen during intermittent bouts of contraction were consistent with previous observations in rats and cats (16, 17, 35). The RSNA response to intermittent contraction was significantly less in the Pro rats than that in the Di rats at the equivalent tension development (Fig. 5).
Effect of estradiol topically applied to the lumbosacral spinal cord on continuous contraction-evoked reflex responses.
In ovariectomized rats (N = 9, 267 ± 9 g body wt), 17β-estradiol topically applied to the lumbosacral spinal cord had no significant effects on baseline MAP, HR, and signal-to-noise ratio for the RSNA before 30-s continuous contraction of the hindlimb skeletal muscle (Table 2). This muscle stimulation increased RSNA, MAP, and HR, and the sympathoexcitatory, pressor, and tachycardia responses were significantly reduced by 17β-estradiol at the equivalent level of muscle tension generated by contraction (Table 2). Plasma estrogen concentrations in ovariectomized rats (N = 8) were 12.5 ± 2.0 pg/ml.
We investigated the effect of the estrous cycle in female rats on the exercise pressor reflex responses. Observations of vaginal smear determined the estrous cycle phase of the rats, and the enzyme-linked immunosorbent assay experiment showed higher concentrations of plasma estradiol in the Pro rats than in the Di rats. In the present experiments to observe the exercise pressor reflex responses, we employed three maneuvers to stimulate muscle in the decerebrate rats. The first was 30-s continuous static contraction of the hindlimb skeletal muscle, commonly used to examine physiological roles of the exercise pressor reflex (16, 33). This maneuver excites both mechanically and chemically sensitive muscle afferents (10, 14, 36). A new finding of the present study was that RSNA and pressor responses to continuous contraction were less in the Pro rats than those in the Di rats. This result indicates that the exercise pressor reflex function fluctuates with the estrous cycle in rats.
Chemically sensitive muscle afferent-mediated sympathoexcitation has been suggested to fluctuate with the ovarian cycle in women (4). In the present study, the second and third maneuvers to stimulate muscle were employed to determine whether muscle mechanoreflex-mediated sympathoexcitation fluctuates with estrous cycle in rats. The second maneuver, passive stretch of Achilles tendon, allowed us to selectively activate mechanically sensitive muscle afferents because it does not generate any metabolites in the muscle (34). Nevertheless, we need to note that the discharging manner of mechanically sensitive group III muscle afferent fibers during stretch is different from that seen during contraction (10). We found that RSNA and pressor responses to passive stretch of Achilles tendon were less in the Pro rats. The third maneuver was 1-min intermittent bouts of static contraction. A brief period (1 s) of contraction is considered to predominantly activate the mechanical component of the exercise pressor reflex (16, 17, 23, 35). This statement is supported by the present observation that the RSNA response to intermittent contraction was rapid in onset and synchronized with the developed muscle tension (Figs. 4 and 5). We found that the synchronized RSNA response with tension development during the intermittent bouts of contraction was less in the Pro rats than that in the Di rats. The data collected in the experiments employing either passive stretch or intermittent contraction demonstrate that the mechanical component of the exercise pressor reflex is suppressed during the proestrus phase compared with that during the diestrus phase in rats. We suggest that the muscle mechanoreflex function in rats fluctuates with the estrous cycle.
As stated, previous studies have demonstrated attenuating effects of estrogen on the pressor response to skeletal muscle contraction in rats and cats (28–31). We confirmed and extended this finding by showing that, in ovariectomized female rats, the RSNA response to skeletal muscle contraction was reduced by estrogen topically applied to lumbosacral spinal cord. These findings suggest that estrogen suppresses the sensitivity of spinal dorsal horn neurons to skeletal muscle afferent input, and that the desensitized dorsal horn neuronal cells are part of the reduced exercise pressor reflex responses.
Even though spinal estrogen application reduced the exercise pressor reflex in the ovariectomized rats in the present study, we did not test if this application would also reduce the responses in gonadally intact rats. It is recognized that chronic estradiol deficiency may induce physiological changes not seen with cyclic fluctuations of estradiol. Thus it would be interesting in future experiments to determine whether spinal estrogen application in diestrus animals reduces the exercise pressor reflex to a larger degree than that in proestrus animals.
The dorsal horn of the spinal cord is the first synaptic site in the exercise pressor reflex arc. Previous literature has provided some potential clues for how estrogen modulates the exercise pressor reflex at the dorsal horn. As stated, the attenuating effects of estrogen in the spinal cord on this reflex have been suggested to be through opioid-dependent mechanisms (30). Other pathways that may contribute to the estrogen's effects include spinal purinergic 2X (P2X) receptors. In cultured rat dorsal root ganglion cells, estrogen has been shown to inhibit ATP-induced increases in intracellular calcium concentration through activated P2X receptors and then voltage-gated calcium channels (2). P2X receptors located in the spinal dorsal horn have been determined to contribute to generation of a pressor response to skeletal muscle contraction (6, 19). Estrogen may block or desensitize spinal P2X receptors. Furthermore, estrogen has been reported to facilitate the release of neuropeptide Y in central nervous system of rats (18), and this peptide reportedly reduced the release of substance P in the cat superficial dorsal horn when unmyelinated primary afferents were excited by electrical stimulation of the tibial nerve (4). Substance P has been determined as spinal neurotransmitters/modulators participating in the exercise pressor reflex generation (15, 37). Although speculative, estrogen may suppress the release of substance P onto the dorsal horn neurons through an effect on central neuropeptide Y. During the estrous cycle in rats, these pathways mediated by estrogen may be involved in the fluctuation of the exercise pressor reflex function.
Supraspinal cardiovascular pathways are also likely involved in the fluctuation of the exercise pressor reflex during the estrous cycle. Estrogen acting on the central nervous system has been reported to have important effects on cardiovascular regulation. For instance, estrogen receptors-α/β are distributed in rat rostral ventrolateral medulla (32), and microinjection of 17β-estradiol into the rat rostral ventrolateral medulla has been shown to reduce RSNA (27). Hayes et al. (9) reported that, in decerebrate and paralyzed cats, intravenous injection of estrogen reduced cardiovascular and ventilator responses to activation of central command, a parallel activation of neural circuits in the brain stem that control motor and cardiorespiratory function during exercise, by electrical stimulation of the mesencephalic locomotor region. Therefore, estrogen in supraspinal cardiovascular pathways is thought to play a role in inhibiting sympathoexcitation. Moreover, it is known that the exercise pressor reflex function is suppressed in central nervous system by the arterial and carotid baroreflex input (25, 26). A possibility exists that reduced exercise pressor reflex responses seen during the proestrus phase were not evoked by the suppressed reflex originating from active muscle, but were due to an enhanced inhibitory effect of the baroreflex mechanism on sympathoexcitation. Goldman et al. (8) have reported that, in rats, RSNA baroreflex sensitivity becomes enhanced during the proestrus phase compared with the diestrus phase. However, they also showed that, while baroreflex-mediated sympathoexcitation was enhanced during the proestrus phase, baroreflex-mediated sympathoinhibition was not different between these phases (8). Therefore, since baroreflex sensitivity to increased arterial pressure is not affected by phase of the estrus cycle, altered baroreflex responses are not likely to contribute to the reduced exercise pressor reflex responses during the proestrus phase. Nevertheless, future studies are necessary to determine the effect of the estrous cycle on functional interactions between baroreceptor and muscle receptor reflexes.
In conclusion, we found that the exercise pressor reflex function fluctuates with the estrous cycle in rats. We also found that the mechanical component of the exercise pressor reflex fluctuates with the estrous cycle. Estrogen may cause these fluctuations through its attenuating effects on the spinal component of the exercise pressor reflex arc.
This work was supported by Japan Society of the Promotion of Science, Grant-in-Aid for Young Scientists B-22790226 (S. Koba) and Akaeda Medical Research Foundation Grant (S. Koba).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: S.K. conception and design of research; S.K., K.Y., and M.M. performed experiments; S.K., K.Y., S.F., and M.M. analyzed data; S.K., K.Y., S.F., and T.W. interpreted results of experiments; S.K. prepared figures; S.K. drafted manuscript; S.K. and T.W. edited and revised manuscript; S.K., K.Y., S.F., M.M., and T.W. approved final version of manuscript.
We thank Dr. Jenni McCord for critical reading of the manuscript.
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