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Division of Cardiovascular Medicine, Departments of Internal Medicine and Human Physiology, University of California, Davis, California 95616
Submitted 11 April 2003 ; accepted in final form 10 June 2003
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ABSTRACT |
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-estradiol needed to attenuate the exercise pressor reflex in male decerebrate cats was 10 µg/ml (Schmitt PM and Kaufman MP. J Appl Physiol 94: 1431-1436, 2003). The threshold concentration for female cats, however, is not known. Consequently, we applied 17-estradiol to a well covering the L6-S1 spinal cord in decerebrate female cats. The exercise pressor reflex was evoked by electrical stimulation of the L7 or S1 ventral root, a maneuver that caused the hindlimb muscles to contract statically. We found that the pressor response to contraction averaged 38 ± 7 mmHg before the application of 17-estradiol (0.01 µg/ml) to the spinal cord, whereas it averaged only 23 ± 4 mmHg 30 min after application (P < 0.05). Recovery of the pressor response to contraction was not obtained for 2 h after application of 17-estradiol. Application of 17-estradiol in a dose of 0.001 µg/ml had no effect on the exercise pressor reflex (n = 5). We conclude that the concentration of 17-estradiol required to attenuate the exercise pressor reflex is 1,000 times more dilute in female cats than that needed to attenuate this reflex in male cats. autonomic nervous system; static contraction; sex hormones; blood pressure; neural control of circulation; women
In animals, the effects of central command and the exercise pressor reflex on the cardiovascular responses to exercise can be examined separately. Nevertheless, there are only two studies examining the effect of estrogen on the cardiovascular responses to exercise, and both were done in male decerebrate cats. The first found that intravenous injection of estrogen attenuated central command but did not attenuate the exercise pressor reflex (9). The second found that spinal application of 17-estradiol attenuated the exercise pressor reflex only at a concentration exceeding the physiological range (20). These findings were surprising because estrogen receptors have been reported to be located on many neurons in the dorsal horn of the spinal cord (1). Moreover, many neurons displaying estrogen receptors are found in laminae of the dorsal horn that are known to receive synaptic input from group III and IV muscle afferents (4, 14). These findings raised the possibility that the attenuation of the exercise pressor reflex by estrogen was gender specific. In the present study, we assessed this possibility and attempted to identify the spinal cord as one location for this gender-specific effect. Specifically, we found that in female cats, spinal application of 17-estradiol attenuated the pressor reflex response to static contraction at a concentration that was 1,000 times lower than that needed to attenuate this reflex response in males.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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A laminectomy was performed to expose the lumbar and sacral spinal cord. The L7 or S1 ventral root was identified and cut. A well of vinyl polysiloxane (VPS; Jeneric/Pentron) was installed on the spinal cord, enclosing the dorsal roots of L6-S1. The method has been described in detail elsewhere (2, 25). To ensure its integrity, the well was filled with saline and checked for leakage. The well was filled either with saline or drugs dissolved in oil throughout the experiment. The skin of the back was used to form a space for a pool around the exposed parts of the spinal cord, including the VPS well, and filled with warm mineral oil (37°C). The saline-containing well, therefore, was seated in a mineral oil pool; this allowed us to continuously monitor the well for leaks. Only data derived from cats in which no leakage was detected were analyzed.
The musculature of the left hindlimb was exposed, and all visible branches of the sciatic nerve except for those innervating the triceps surae muscles were sectioned. The calcaneal bone was severed, and its tendon was attached to a force transducer (model FT 10, Grass). The knee was clamped in place.
The cat was removed from the ventilator and was allowed to breathe room air spontaneously. Airflow was measured with a Pneumotach (Fleisch) attached in series to the trachea tube. Airflow was integrated (Gould), breath by breath, to yield tidal volume, which was used to calculate minute ventilation.
Determination of 17-estradiol plasma levels. Arterial blood samples, taken from the carotid catheter, were collected in EDTA-coated tubes before and 60 min after 17-estradiol was applied to the spinal cord. The samples were kept on ice for no more than 1 h before they were centrifuged for 5 min at 4,000 rpm and at 4°C. The plasma samples were stored at -80°C until further processing. 17-Estradiol was extracted in diethyl ether, and the plasma concentration was measured by using a radioimmunoassay (21). The detection limit was 6 pg/ml.
Protocols. Static contractions were evoked by electrically stimulating the L7 or S1 ventral root at 2-3 times motor threshold (0.1 ms, 30-40 Hz). Resting tension of the triceps surae muscles was set at 1.0 kg. The contraction period was 60 s. Each cat underwent two to three static contractions before a blood sample for the analysis of plasma 17-estradiol was taken and drugs were administered. The time between subsequent electrical stimulations was 15 min. 17-Estradiol, which was dissolved in mineral oil, was placed in the spinal VPS well in concentrations of 0.001, 0.01, or 0.1 µg/ml. The volume was 200 µl. In a previous study in rats (2), the tissue concentration of neurokinin A (molecular weight 1,133) in the underlying superficial laminae of the spinal cord (at a depth of 500 µm) was 25-70 times lower than the concentration in the superfusate in the well. Peak concentration was reached after 30 min of perfusion. The tissue concentration in the deeper laminae (depth 0.75-1.5 mm) was found to be lower than that at the superficial laminae (2). In cats, the GABA-blocker muscimol (molecular weight 114-195) applied in a similar manner to the dorsal root of the spinal cord, was found to block the exercise pressor reflex (25). Therefore, we choose our dosages to be in the range of 70 times the physiological estrogen concentration in blood during peak estrus.
Each cat received only one of the concentrations of 17-estradiol. The solution remained in the well for 60 min, and the responses to the exercise pressor reflex were tested 30, 45, and 60 min after the estrogen solution had been topically applied. After 60 min, a second arterial blood sample for the analysis of plasma 17-estradiol was taken, and the estradiol-oil solution was removed from the well. The well was flushed three times and then filled with saline to determine whether the exercise pressor reflex would recover in a 2-h time period.
Data analysis. The data reported in this study was obtained from female cats in which the presence of ovaries was confirmed postmortem. Values for mean arterial pressure, heart rate, and minute ventilation are expressed as means ± SE. Baseline MAP and heart rate were recorded immediately before the muscle contraction; peak values represent the highest level reached during the muscle contraction. Ventilation was calculated as a minute volume immediately before (baseline) and during the contraction (peak). Statistical significance was determined by one- and two-way repeated-measures ANOVA, followed by Dunnett's and Tukey's post hoc tests when applicable. The criterion for statistical significance was P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-estradiol on the pressor response to muscle contraction. 17-Estradiol applied topically to the spinal cord in a concentration of 0.01 and 0.1 µg/ml significantly attenuated the pressor response to contraction of the triceps surae muscles (Tables 1 and 2; Figs. 1 and 2). For the larger of the two concentrations (i.e., 0.1 µg/ml) the attenuation started after a latent period of 45 min, whereas for the smaller concentration (i.e., 0.01 µg/ml) it started after a latent period of 30 min (Table 2, Fig. 2). The pressor response to muscle contraction did not appear to recover for 2 h after the removal of 17-estradiol from the spinal cord in any of the cats. Baseline mean arterial pressure increased moderately in the groups that received 17-estradiol in concentrations of 0.01 and 0.1 µg/ml (Figs. 3 And 4). For the group receiving a concentration of 0.01 µg/ml, mean arterial pressure increased transiently from 101 ± 7 to 114 ± 6 mmHg (P < 0.05). Similarly, for the group receiving a concentration of 0.1 µg/ml, mean arterial pressure increased from 130 ± 12 to 145 ± 10 mmHg (P < 0.05). The lowest concentration (i.e., 0.001 µg/ml) of 17-estradiol tested had no effect on the pressor response to contraction and no effect on baseline mean arterial pressure (Table 2). None of the three concentrations of 17-estradiol, applied topically to the spinal cord, increased the concentrations of this hormone in the arterial blood (Table 3).
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Effects of 17-estradiol on the cardioaccelerator and ventilatory responses to muscle contraction. Spinal application of 17-estradiol in the concentrations tested had no effect on the cardioaccelerator response to contraction (Table 2; Figs. 1, 3, and 4). Similarly, spinal application of 17-estradiol had no effect on the ventilatory response to contraction. Baseline minute ventilation increased over time in both groups. Baseline heart rate increased in the group that received a concentration of 0.1 µg/ml 17-estradiol (P < 0.05; Fig. 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-estradiol, topically applied to the lumbosacral spinal cord, attenuated the reflex pressor response to static contraction of the triceps surae muscles. The threshold concentration of 17-estradiol (i.e., 10 ng/ml) needed to achieve this attenuation in female decerebrate cats was 1,000 times more dilute than the threshold concentration needed to attenuate the reflex pressor response to contraction in male decerebrate cats (20). In our experiments, topical application of 17-estradiol in any of the concentrations tested had no effect on the cardioaccelerator and ventilatory responses to contraction.
In humans, women have been reported to generate smaller pressor and muscle sympathetic nerve activity responses to exercise than men (7, 8). The difference has been attributed to the effect of 17-estradiol on the exercise pressor reflex (7, 8). Our present findings combined with those reported previously appear consistent with the reported effect of 17-estradiol in humans. Moreover, our findings suggest that the site of action of this sex hormone was limited to the L6-S1 spinal cord. In particular, this site of action might be the superficial laminae of the L6-S1 dorsal horn, both of which receive synaptic input from group III and IV hindlimb muscle afferents (4). Alternatively or in combination with the dorsal horn, the site of action might be the L6-S1 dorsal root ganglia, which contain the cell bodies of the group III and IV afferents evoking the exercise pressor reflex (13).
We did not measure the tissue concentration of 17-estradiol after applying the hormone to the spinal cord and consequently can only speculate about its concentration in the dorsal horn. This speculation is based on the spinal distribution of neurokinin A (2), which varies markedly from 17-estradiol in both its molecular weight and its lipid solubility. Consequently, our speculation about the concentration of 17-estradiol in the dorsal horn of the spinal cord must be viewed cautiously. On the basis of the diffusion of neurokinin A through the dorsal horn, the lowest effective concentration (i.e., 0.01 µg/ml) of 17-estradiol 500 µm below the dorsal surface of the spinal cord would have been about twice its peak plasma concentration during estrus in cats (23).
Our present and previous findings suggest that the 17-estradiol-induced attenuation of the pressor reflex response to static contraction in cats is sex specific. Little is known about the gender-specific distribution of estrogen receptor in the spinal cord. In female rats (19) and cats (22), however, both the L6 and S1 dorsal horn contain estrogen receptors. The superficial dorsal horn (i.e., laminae I and II) has been shown to contain more estrogen receptor-
than estrogen receptor- (19). This portion of the dorsal horn is particularly relevant to the interpretation of our findings because 17-estradiol was topically applied to the dorsal surface of the spinal cord. Laminae V, the site where many thin fiber muscle afferents synapse (4), also contains estrogen receptor- and estrogen receptor-, with the latter appearing to be more plentiful than the former (19). In addition, cells of the L6-S1 dorsal root ganglia have been shown to contain estrogen receptors, with some containing only estrogen receptor-, others containing only estrogen receptor-, and still others both estrogen receptor- and estrogen receptor- (18). Moreover, dorsal root ganglia cells staining positive for estrogen receptors were either medium or small in size; in addition, some stained positive for the vanilloid receptor 1 protein, which binds capsaicin (18). Cells fitting this profile are thought to give rise to group III (i.e., A
) and IV (i.e., C) fibers, which comprise the afferent arm of the exercise pressor reflex arc.
The sex-specific effect of estrogen in the spinal cord might also originate from differences in the intracellular pathways and downstream events caused by the stimulation of either intracellular or membrane-bound estrogen receptors. For example, female mice possess a sex-specific analgesic mechanism, which is induced by stress and is not dependent on either opioids or N-methyl-D-aspartate receptors. This analgesic mechanism, however, is dependent on estrogen, and it has been attributed to a gene that is turned off by testosterone exposure during sexual differentiation (16). A similar sex-specific mechanism may be responsible for our finding that the threshold concentration of 17-estradiol needed to attenuate the exercise pressor reflex was 1,000 times more dilute in female cats than it was in male cats.
Estrogen exerts its effects by means of two mechanisms, the first of which is genomic and the second is nongenomic. For the genomic mechanism, estrogen penetrates the cell membrane and subsequently binds to intracellular receptors that in turn regulate gene expression. For the nongenomic mechanism, estrogen binds to membrane receptors that modulate either ion channels or second messengers (11). For example, 1 µM 17-estradiol has been shown in vitro to rapidly inhibit high-voltage calcium channels in rat dorsal root ganglion cells (12). The genomic mechanism has a latency of 
30 min, whereas the nongenomic mechanism has a latency of seconds (17). At first glance, our findings might appear to fit the genomic mechanism of action because spinal application of 17-estradiol attenuated the pressor response to static contraction of the triceps surae muscles after a latent period of 30-45 min. Much of this latent period, however, might have been caused by the diffusion of 17-estradiol through the dorsal horn. Consequently, we hesitate to attribute the estrogen-induced attenuation of the pressor reflex to either mechanism.
We found that topical application of 17-estradiol to the spinal cord did not increase the concentration of this hormone in the arterial blood. Moreover, the concentrations of 17-estradiol that we measured in the arterial blood were similar to those reported previously in nonpregnant female cats (23). Consequently, the probability that 17-estradiol circulated to other central neural structures to exert its attenuating effect on the exercise pressor reflex is low. The probability is also low that 17-estradiol traveled to the thoracic and upper lumbar cord to affect the sympathetic outflow because the VPS well restricted its access to the lower lumbar and sacral cord.
Although static contraction significantly increased mean arterial pressure and heart rate in our experiments, it had no effect on ventilation. This lack of an effect on ventilation was probably due to the fact that the average peak tension developed by the contracting triceps surae muscles in our studies was less than half that of maximum. Similar responses to moderate to small contractions have been reported previously in cats (26). The ventilatory response to contraction was probably the sum of two competing reflex mechanisms, the first of which is an inhibitory influence arising from neuronal circuitry in the spinal cord (5); the second is an excitatory influence arising from circuitry in the medulla. In any event, the primary effect of the exercise pressor reflex (15) in both animals and humans is to increase arterial pressure as well as sympathetic outflow to the vasculature of skeletal muscles. This reflex is thought to exert only small effects on heart rate and ventilation (10, 24), which during exercise may be controlled primarily by central command (24).
In summary, we have found that topical application of 17-estradiol to the L6-S1 spinal cord attenuated the reflex pressor response to static contraction of the triceps surae muscles in female decerebrate cats. The concentration of 17-estradiol that caused this attenuation (i.e., 0.01 µg/ml) was 1,000 times more dilute than that needed to attenuate this response in male cats. The concentration of 17-estradiol in the superficial laminae of the dorsal horn was not measured, but it may have approached the physiological range. In any event, our present finding may help to explain the neural mechanism whereby women display a smaller pressor and muscle sympathetic nerve activity response to static exercise than do men (7).
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-oestradiol-induced inhibition of contraction in mammalian vascular smooth muscle. J Physiol 499: 497-511, 1997.-Estradiol inhibits high-voltage-activated calcium channel currents in rat sensory neurons via a non-genomic mechanism. Life Sci 70: 2047-2059, 2002.[Web of Science][Medline]
and - coexist in a subpopulation of sensory neurons of female rat dorsal root ganglia. Neurosci Lett 319: 71-74, 2002.[Web of Science][Medline]
- and -immunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal cord. Cell Tissue Res 304: 193-214, 2001.[Web of Science][Medline]
-estradiol attenuate the exercise pressor reflex in male cats. J Appl Physiol 94: 1431-1436, 2003. concentrations in plasma: relation to estrous behavior and cornification of exfoliated vaginal epithelium. Biol Reprod 21: 953-963, 1979.[Abstract]
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) between the peak response to contraction and its corresponding baseline value. A: 0.1 µg/ml 17
E, minute ventilation. *Responses to contraction that differ significantly (P < 0.05) from their corresponding baselines. bBaseline value is significantly greater (P < 0.05) than the corresponding control baseline value.