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Division of Cardiovascular Medicine, Departments of Internal Medicine and Human Physiology, University of California, Davis, California
Submitted 27 July 2004 ; accepted in final form 20 September 2004
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ABSTRACT |
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-estradiol (0.01 µg/ml) with and without the addition of 1,000 µg naloxone, a µ- and 
-opioid antagonist, to a spinal well covering the L6–S1 spinal cord in decerebrated female cats that were either gonadally intact or ovariectomized. 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, in gonadally intact cats, the attenuating effect of estrogen was more pronounced than that in ovariectomized cats. We also found that, in gonadally intact female cats, naloxone partly reversed the attenuation of the pressor response to static contraction caused by spinal estrogen application. For example, in intact cats, the pressor response to contraction before estrogen application averaged 39 ± 4 mmHg (n = 10), whereas the pressor response 60 min afterward averaged only 18 ± 4 mmHg (P < 0.05). In contrast, the pressor response to contraction before estrogen and naloxone application averaged 33 ± 5 mmHg (n = 11), whereas afterward it averaged 27 ± 6 mmHg (P < 0.05). In ovariectomized cats, naloxone was less effective in reversing the attenuating effect of estrogen on the exercise pressor reflex. sex hormones; static contraction; blood pressure; neural control of circulation; autonomic nervous system
The mechanism by which spinally applied estrogen attenuated the reflex pressor response to static contraction is unknown. Nevertheless, µ-opioid receptors and -opioid receptors have been implicated in the spinal regulation of the exercise pressor reflex in cats (8, 11, 18). Specifically, intrathecal application of µ-opioid and -opioid receptor agonists to the lumbar spinal cord attenuated the reflex pressor response to static contraction. Moreover, naloxone, a µ-opioid and -opioid antagonist, inhibited this attenuation (11, 18). These findings suggest that opioids are candidates to mediate the spinal action of estrogen on the exercise pressor reflex.
Further evidence for an interaction between opioids and estrogen in the spinal pathways mediating the exercise pressor reflex arc comes from studies on pain. The thin fiber afferents (i.e., group III and IV) believed to comprise the sensory arm of the exercise pressor reflex arc may also be capable of evoking the sensation of muscle pain. Estrogen has been implicated in influencing opioid-mediated analgesia (4, 15, 23). In addition, the activation and expression of opioid receptors have been linked to the presence of estrogen in several different areas of the central nervous system (17, 22). In the present study, we tested the hypothesis that µ- or -opioids mediate the attenuating effect of estrogen on the exercise pressor reflex in gonadally intact female cats as well as in ovariectomized cats. Our study includes data from gonadally intact and ovariectomized female cats treated with estrogen alone or estrogen in combination with naloxone.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The cat was placed in a Kopf stereotaxic and spinal unit and then given dexamethasone (4 mg iv). A midcollicular decerebration was performed, after which anesthesia was terminated. All neural tissue rostral to the section was removed. Bleeding was controlled and the cranial vault was filled with agar (37°C).
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 constructed on the spinal cord, enclosing the dorsal roots of L6–S1. The method has been described in detail elsewhere (2, 29). 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 saline 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, and the well was 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 tracheal cannula. Airflow was integrated (Gould), breath by breath, to yield tidal volume, which in turn was used to calculate minute volume of ventilation.
Protocols. Static contractions were evoked by electrically stimulating the L7 or S1 ventral root at two to three 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 drugs were administered. The time between subsequent contractions was 15 min. Responses to contraction obtained before spinal application of estrogen alone or in combination with naloxone were averaged.
In 6 ovariectomized and in 10 intact cats, water-soluble 17-estradiol was dissolved in 200 µl saline to achieve a final concentration of 0.01 µg/ml. This solution was then applied to the spinal cord by placing it in the VPS well. Eleven gonadally intact female cats and 7 ovariectomized cats were treated with the same estrogen solution as that described above except that 1,000 µg naloxone were added to it. Similarly, this solution was placed in the spinal VPS well.
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 (depth: 0.5 mm) 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). Therefore, we choose our dosage of estrogen to be in the range of 70 times the physiological estrogen concentration in blood during peak estrus. Our previous study showed that an estradiol concentration of 0.01 µg/ml was sufficient to attenuate the exercise pressor reflex in gonadally intact female cats 30 min after topical application to the cord (24). We choose the naloxone dose (1,000 µg) because it was found in a previous study (11) to restore the pressor response to contraction that was attenuated by the -opioid agonist [D-Ala2,Met5-enkephalin].
Each cat received only one of the solutions. The solution remained in the well throughout the experiment and the responses to the exercise pressor reflex were tested 30, 45, and 60 min after the estrogen solution had been applied. The stability of the exercise pressor reflex over this 60-min time period has been established (24, 25). Similarly, estrogen has been shown to attenuate the reflex over this time period (24, 25). At the end of the procedure, cats were euthanized and presence or absence of ovaries was confirmed.
Data analysis. Values for mean arterial pressure, heart rate, and minute ventilation are expressed as means ± SE. Baseline MAP and HR were taken as the steady-state values immediately before static contraction; peak values represent the highest level reached during static 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.
Data derived from ovariectomized cats that received a combination of 17-estradiol and naloxone (n = 7) were compared with data derived from ovariectomized cats treated with 17-estradiol alone (n = 6). The data derived from gonadally intact female cats treated with a combination of 17-estradiol and naloxone (n = 11) were compared with data derived from gonadally intact female cats that had undergone spinal 17-estradiol treatment (n = 10). For this latter group of 10 gonadally intact female cats, the data from 6 were taken from a previously published report (24), and the remaining 4 were taken from newly performed experiments.
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RESULTS |
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-estradiol on the pressor, cardioaccelerator and ventilatory responses to contraction in gonadally intact female cats.
17-Estradiol alone significantly attenuated (P < 0.05) the pressor responses to static contraction (n = 10; Tables 1 and 2, Fig. 1) 30, 45, and 60 min after its application to the spinal cord. Baseline mean arterial pressure did not increase over time (Fig. 2). The cardioaccelerator response was significantly reduced at the 60-min time point of application. The ventilatory response to contraction was unaffected by 17-estradiol treatment in gonadally intact female cats (Table 2). Baseline ventilation did not vary with time, whereas baseline heart rate increased over time from 166 ± 13 to 186 ± 17 mmHg at the 60-min time point (P < 0.05).
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-estradiol in gonadally intact female cats.
The pressor responses to static contraction 30, 45, and 60 min after naloxone and 17-estradiol were applied topically to the surface of the spinal cord (n = 11) were significantly greater (P < 0.05) than the pressor responses to static contraction, respectively, 30, 45, and 60 min after 17 -estradiol alone was applied topically to the surface of the cord (n = 10; Tables 1 and 2, Fig. 1). Baseline mean arterial pressure was unaffected by naloxone applied in combination with 17-estradiol (Fig. 2). Addition of naloxone to the spinal 17-estradiol solution did not affect the cardioaccelerator and ventilatory responses to contraction (Table 2). Baseline heart rate increased significantly (P < 0.05) over time from 187 ± 7 beats/min (pretreatment, n = 11) to 202 ± 6 beats/min (30-min time point, n = 11). Baseline ventilation increased from 628 ± 71 ml/min (pretreatment, n = 11) to 1,165 ± 212 ml/min (60-min time point; n = 11, P < 0.05).
Effects of 17-estradiol on the pressor, cardioaccelerator, and ventilatory response to contraction in ovariectomized cats.
17-Estradiol alone significantly attenuated (P < 0.05) the pressor responses to static contraction (n = 6; Tables 1 and 2, Fig. 1) 45 and 60 min after its application to the spinal cord. Baseline mean arterial pressure did not increase over time (Fig. 3). The cardioaccelerator and ventilatory responses to contraction were unaffected by 17-estradiol treatment in ovariectomized cats (Table 2). Baseline heart rate and ventilation did not vary with time.
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-estradiol in ovariectomized cats.
In contrast to the effect of 17-estradiol alone, the combination of naloxone and 17-estradiol did not significantly attenuate the pressor response to static contraction 45 min after its topical application to the spinal cord (n = 6; P > 0.05; Tables 1 and 2, Fig. 1). However, the combination of naloxone and 17-estradiol still significantly attenuated the pressor response to contraction 60 min after its application (P < 0.05; see Fig. 1). At the 30-min time point, baseline mean pressure increased (from 131 ± 10 mmHg to 156 ± 15 mmHg, n = 7), returning toward pretreatment values at the 45-min time point. Naloxone addition to the spinal 17-estradiol solution did not affect the cardioaccelerator and ventilatory responses to static contraction (Table 2). However, baseline heart rate increased significantly (P < 0.05) over time from 147 ± 11 beats/min (pretreatment, n = 7) to 161 ± 11 beats/min (n = 7) and 162 ± 14 beats/min (n = 7) at the 30- and 60-min time points, respectively. Baseline ventilation did not change over time. |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-estradiol was applied to the spinal cord. This attenuation was partially reversed by the opioid blocker, naloxone. Our findings suggest that estrogen acted in the spinal cord through a µ- and/or -opioid-dependent mechanism to attenuate the pressor response to contraction. Nevertheless, not all the gonadally intact cats tested showed equal sensitivity toward naloxone treatment. Specifically five responded to naloxone treatment with a complete loss of the estrogen effect, whereas six others responded to naloxone by reducing their responses by one-third. This may indicate that opioids account partially for estrogen’s attenuating effect on the pressor response to contraction, with other mechanisms making a contribution as well.
In ovariectomized cats, spinal application of 17-estradiol alone attenuated the pressor response to static contraction after 45 and 60 min, but it did not attenuate the responses after 30 min. This latter observation contrasts with that made in gonadally intact cats where the pressor response to contraction was attenuated by 17-estradiol alone after 30 min as well as after 45 and 60 min. The difference in onset for the 30-min time point might be explained by the possibility that the low estrogen levels in ovariectomized cats do not activate estrogen-dependent pathways as frequently as they do in gonadally intact female cats undergoing cyclic changes of their hormone profile. The lack of use of cellular mechanisms involving these estrogen-dependent pathways might result in the downregulation of either estrogen or opioid receptors. For example, the presence of estrogen in ovariectomized rats has been reported to either up- or downregulate the neuronal estrogen receptors in a region-specific manner (12, 26). Long-term estrogen treatment of ovariectomized rats decreased levels of estrogen receptor-
mRNA in the dorsal root ganglion, whereas this treatment increased levels of estrogen receptor- (27). This last finding raises the possibility that the estrogen-dependent mechanism that we found in gonadally intact female cats involved estrogen receptor-. In addition, the estrogen-dependent mechanism that we found in ovariectomized cats may have also involved estrogen receptor- to some small extent, or it may have involved a compensating mechanism, which at this time is unknown.
We also found that naloxone was less effective in preventing the magnitude of the attenuating effect of 17-estradiol on the pressor response to contraction in ovariectomized cats than it was in preventing the magnitude of the attenuating effect in gonadally intact cats. We can offer no explanation for this finding, but it is obviously related to the effects of estrogen on feline development. The explanation probably involves the effects of ovariectomy on the densities and affinities of opioid receptors in the spinal cord. To our knowledge, the only relevant literature on this issue is the finding that ovariectomy in mice has been shown to reduce the density of µ-opioid receptors in the hypothalamus of mice (13).
Estrogen is thought to affect neuronal µ-opioid pathways by two distinct mechanisms (14). First, estrogen rapidly activates a protein kinase A pathway in -endorphin-containing neurons, leading to the uncoupling of µ-opioid receptors from inwardly rectifying potassium channels. We assume that the first mechanism is probably not the one by which estrogen exerts its attenuating effect on the exercise pressor reflex because its activation results in reduced hyperpolarization and increased excitability of neurons involved. This assumption was supported by a recent study that described a gender difference in the contribution of inwardly rectifying potassium channels to opioid analgesia (20). Specifically, µ-opioid antinociception in female mice was independent of inwardly rectifying potassium channels, whereas in male mice this antinociception was dependent on these channels.
Second, estrogen may act by initiating a cascade of events, which has its final effect on a group of neurons insensitive to estrogen. Specifically, estrogen may reduce the effectiveness of opioid (e.g., -endorphin) binding to µ-opioid receptors (14). Similarly, estrogen may reduce ligand binding to GABA receptors (14). These effects alone will result in reduced opioid effects and GABA effects in cells carrying receptors to these agonists. However, both events also decrease the inhibitory influence of µ-opioid receptors and GABA receptors on further -endorphin release, making more -endorphin available to other neurons. Consequently, more -endorphin can bind to opioid receptors located on neurons not equipped with estrogen receptors. Therefore, estrogen-insensitive neurons but not estrogen-sensitive neurons may experience amplification of opioid-induced inhibition. To the extent that these neurons are a component of the exercise pressor reflex arc, they may be the source of the estrogen-induced attenuation of the pressor response to static contraction reported in our experiments.
In addition to its rapid effects on neuronal opioid pathways, estrogen can exert its effects through genomic mechanisms, which modulate the density of opioid receptors in selective brain areas (16, 17). These mechanisms involve the binding of estrogen-response elements to target genes, or protein-protein interaction subsequently modifying transcription. The time necessary for new protein synthesis is 
30–45 min, and probably an even much longer time is required to alter cellular physiology (1). It is unlikely, therefore, that the effects of estrogen seen in our study were caused by the activation of a genomic mechanism.
The variability of the pressor responses to static contraction after spinal application of 17-estradiol and naloxone in gonadally intact female cats may possibly be related to differences in their estrous cycles. Cats are seasonal polyestrous, and experience during spring and summer high incidence of estrus, which will lead to ovulation only if coitus occurs. Otherwise, another short cycle of proestrus and estrus will follow a time of interestrus (7). Previously we found that it was not possible to identify these brief periods of estrogen peaks (50–80 pg/ml) by measuring 17-estradiol in the plasma (24). Consequently, we did not attempt to make these measurements in this study. Nevertheless, although no obvious relationship existed between susceptibility to naloxone and the date that we studied the cats, the possibility remains that variability of the hormonal status modified the response to naloxone. The fact that the results observed in ovariectomized cats were relatively homogenous strengthens this hypothesis. Five of seven ovariectomized cats showed the absence of any effect of naloxone 60 min after its application.
Another source of variance might be the activation of the 
-opioid system. In the rat, the -opioid receptor density in laminae I and II of the lumbosacral spinal cord has been found to be elevated during proestrus/estrus (10). A complex synergistic interaction has been shown to occur among the different spinal opioid systems that may account for pregnancy nociception (5, 9). The thin fiber muscle afferents (group III and IV) believed to compromise the sensory arm of the exercise pressor reflex arc is also thought to signal nociceptive stimuli. However, an early study in halothane-anesthetized cats suggested that -opioids are not directly involved in the neural pathways of the exercise pressor reflex (8). Specifically, application of a -opioid receptor agonist failed to attenuate the spinal release of substance P in response to sciatic nerve stimulation at current intensities that activated A- and C (i.e., group III and IV) fibers and resulted in a blood pressure increase.
The question arises whether naloxone itself may interfere with the exercise pressor reflex through an estrogen-independent pathway, which would make our results difficult to evaluate. Although naloxone binds preferably to µ-opioid receptors, it will also bind to - and -opioid receptors. In addition, naloxone has been shown to have either excitatory or inhibitory effects on the discharge of dorsal horn neurons receiving nociceptive input (3). Both of these factors can make the data resulting from its use difficult to interpret. This might especially be the case if there is a tonic release of endogenous ligands acting on two or more opioid receptors. We think these possibilities are unlikely because a previous study from our laboratory, which used cats of both genders, showed that intrathecal application of naloxone in the concentration used in the present study had no effect on either baseline values or pressor, cardioaccelerator, or ventilatory responses to static contraction (11). These results may at first glance seem confusing, because one might expect to see an effect of naloxone in gonadally intact female cats. However, the group of animals studied was small (n = 5) and did not control for gender or estrous cycle. Consequently, an estrogen-dependent effect of naloxone may have been masked.
The muscle tension developed during contraction varied between the two treatment groups of ovariectomized animals (3.8 ± 0.3 vs. 5.8 ± 0.7 kg). This raises the concern that the difference in muscle tension may have affected the magnitude of the reported pressor responses (27 ± 5 vs. 31 ± 8 mmHg, pretreatment). However, compared with the data (39 ± 4 vs. 33 ± 5 mmHg, pretreatment) obtained in gonadally intact female cats, which developed a more similar muscle tension (5.7 ± 0.6 vs 5.2 ± 0.6 kg), the difference in the blood pressure response of the two treatment groups of ovariectomized animals appears minimal and within the range of physiological variability. We argue that this observation reduces the likelihood for the absence of an attenuating effect in naloxone treated ovariectomized cats being caused by high muscle tension.
In decerebrate cats, the ventilatory response to contraction appears to be the result of two competing mechanisms. The first is an inhibitory influence arising from neuronal circuitry in the spinal cord, and the second is an excitatory influence arising from neuronal circuitry in the medulla (6). The first mechanism appeared on average to be the dominant one in our experiments, but in individual instances the second mechanism appeared quite powerful. Whatever the dominant mechanism, ventilation in decerebrate cats displays a high degree of variability that in turn makes it difficult to draw any conclusions about the effect of spinally applied estrogen on the ventilatory response to contraction.
The vasoconstrictor response to exercise is believed by most investigators to be primarily under the control of the exercise pressor reflex, whereas the ventilatory response to exercise is believed to be primarily under the control of central command (21, 28). The cardioaccelerator response to exercise may be under the control of both neural mechanisms, but in the decerebrated unanesthetized cat the heart rate response to static contraction is often small and variable. Consequently, we were not surprised that spinal application of 17-estradiol did not attenuate on a statistical basis the ventilatory and cardioaccelerator responses to static contraction in most of our experiments. This might especially be the case because the sample size was relatively small.
In summary, we found that, in gonadally intact female cats, estrogen exerted its attenuating effect on the exercise pressor reflex at least in part by activating opioid pathways in the spinal cord. We also found that, in ovariectomized cats, estrogen exerted its attenuating effect on this reflex by activating opioid pathways, but the role played by opioids in the estrogen-induced attenuation of the reflex was less pronounced than it was in gonadally intact cats. We had no knowledge about the timing of the ovariectomy performed on the cats used in this report. Nevertheless, we speculate that the effect of ovariectomy on our findings is attributable to changes in the number and affinities of estrogen and opioid receptors in the spinal cord caused by the absence of estrogen.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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and "mixed action" agonists. Drug Alcohol Depend 63: 215–228, 2001.[CrossRef][Web of Science][Medline]
2-adrenergic analgesia and analgesic sex differences. Proc Natl Acad Sci USA 100: 271–276, 2003.-opioid agonist inhibition in response to uterine cervical distenstion. Anesthesiology 96: 375–380, 2002.[CrossRef][Web of Science][Medline]
-estradiol attenuate the exercise pressor reflex in male cats. J Appl Physiol 94: 1431–1436, 2003.This article has been cited by other articles:
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  P. M. Schmitt, K. Gohil, and M. P. Kaufman Spinal estrogen attenuates the exercise pressor reflex but has little effect on the expression of genes regulating neurotransmitters in the dorsal root ganglia J Appl Physiol, March 1, 2006; 100(3): 958 - 964. [Abstract] [Full Text] [PDF]
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MAP), cardioaccelerator responses (
E) to static contraction before (pretreatment) and after (30, 45, and 60 min) 17
