Journal of Applied Physiology
Vol. 81, No. 5, pp. 1911-1916, November 1996
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

Dextromethorphan affects ventilation differently in male and female rats

Evelyn H. Schlenker

Department of Physiology and Pharmacology, University of South Dakota School of Medicine, Vermillion, South Dakota 57069

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Schlenker, Evelyn H. Dextromethorphan affects ventilation differently in male and female rats. J. Appl. Physiol. 81(5): 1911-1916, 1996.Subcutaneous administration of aspartic acid results in a long-lasting but reversible depression of ventilation in male but not in female rats. Aspartic acid acts on N-methyl-D-aspartate receptors. The present study tested the hypothesis that a noncompetitive N-methyl-D-aspartate-receptor antagonist, dextromethorphan (Dex), would depress ventilation in female rats and stimulate it in male rats. Moreover, Dex administered prior to aspartic acid should prevent the aspartic acid-induced depression of ventilation in male rats. In female rats, Dex caused a 30% depression of ventilation relative to saline at 5 and 10 mg/kg (P < 0.01) but not at the highest dose (20 mg/kg). In male rats, Dex had no effect on ventilation. At a dose of 20 mg/kg, Dex depressed oxygen consumption to 50% of the saline value at all time points in female rats (P < 0.001) and in male rats 45 and 60 min after administration. The time points when Dex depressed ventilation and oxygen consumption were different in female rats, suggesting that the depression of ventilation was not the result of a depression in oxygen consumption. During a hypercapnic challenge (7% CO₂), female rats treated with 5 and 10 mg/kg of Dex exhibited a smaller increase in ventilatory response relative to saline treatment. At a dose of 20 mg/kg, the hypercapnic responsiveness of male rats was markedly stimulated (85.8 ± 8.95 ml/min) relative to saline (50.6 ± 9.14 ml/min; P < 0.001). Finally, Dex administered before aspartic acid prevented the aspartic acid-induced depression of ventilation in male rats. Thus, in rats, Dex has gender-specific effects on ventilation and these effects are not associated with changes in oxygen consumption.

sex; oxygen consumption; N-methyl-D-aspartate receptor

INTRODUCTION

THE ROLE of N-methyl-D-aspartate (NMDA) receptors in modulating the control of breathing has been well recognized in various species (4, 11, 12, 19). Moreover, Connelly et al. (9) demonstrated that NMDA receptors affected ventilation differently in male Sprague-Dawley compared with Wistar rats, suggesting a genetic basis for their effect in the control of breathing. Gender differences associated with NMDA function in the control of breathing have only been studied indirectly. For example, Schlenker and Goldman (22) showed that neonatal treatment of rats with aspartic acid (which acts on NMDA receptors) results in adult male rats that exhibit alveolar hypoventilation and blunted ventilatory responses to hypercapnia. Female rats treated in a similar manner did not exhibit these abnormalities relative to vehicle-treated female rats. In a separate study, these investigators (21) reported that subcutaneous administration of aspartic acid into normal adult rats caused a long-lasting but reversible depression of breathing in male but not in female rats.

With this background, the present study was designed to test the hypothesis that dextromethorphan (Dex), a noncompetitive NMDA-receptor antagonist (7, 8, 26), would cause a depression in breathing in female rats but not in male rats. Furthermore, Dex administered before aspartic acid should prevent the aspartic acid-induced depression of ventilation in male rats. This antagonist was selected because, unlike MK-801 (dizocilpine), Dex does not have the psychotomimetic effects (10, 28). Subcutaneously administered Dex also readily and rapidly enters the central nervous system and has been shown to have much lower toxicological effects compared to MK-801 (3, 29).

Because Dex has not been previously investigated in relation to its effects on the control of breathing or oxygen consumption, the first part of the study consisted of a dose-time-response study in male and female rats. Oxygen consumption was also measured to determine whether Dex had similar effects on both variables, because an increase in ventilation, for example, may have been the consequence of an increase in oxygen consumption. Information from that study was then utilized to determine whether Dex administered before aspartic acid could prevent the aspartic-acid induced depression of ventilation previously noted in male rats (21, 24). Results of these studies will give us more insight into differential mechanisms associated with the NMDA receptor utilized by male and female rats in modulating the control of breathing.

METHODS

Three-month-old male (n = 12) and female (n = 13) Sprague-Dawley rats obtained from Sasco (Omaha, NE) were utilized in these experiments. The rats were housed three to four animals per cage according to gender for at least 1 wk before the commencement of the experiments. During that time, the animals were handled daily and introduced into the plethysmograph (see below) for a period of 30 min/day. Food and water were available ad libitum. Lighting consisted of 12 h on and 12 h off. All procedures were approved by the University of South Dakota Institutional Animal Use and Care Committee.

Ventilation and oxygen consumption in awake animals were determined with the plethysmographic technique used routinely in my laboratory and described in detail previously (21, 23). Briefly, the plethysmographic chamber consisted of a 19-cm-long 9.5-cm-diam Plexiglas cylinder closed at both ends, with ports to allow air or gases (10% oxygen or 7% carbon dioxide) to enter and exit the chamber. The flow rates of gases exiting the chamber were measured with a Gilmont rotameter. The fractional contents of oxygen or carbon dioxide in the air exiting and entering the chamber were measured with a Beckman OM-14 oxygen analyzer and a Vacumed carbon dioxide analyzer, respectively. Pressure changes associated with ventilation were measured with a low-pressure Statham pressure transducer coupled to a Grass 7D recording system. Ventilatory parameters were determined from an average of 20 breaths/exposure period and included tidal volume, frequency of breathing, inspiratory and expiratory times, and ventilation. Oxygen consumption was evaluated with the flow-through system. For this measurement, the difference between the fractional contents of oxygen entering and exiting the chamber was measured and multiplied by the flow rate of air through the chamber.

RESULTS

Ventilation was depressed in female rats with Dex at a dose of 5 mg/kg 15, 30, and 45 min and at a dose of 10 mg/kg 30 min after injection relative to saline (Fig. 1). The depression of ventilation was the result of a decreased frequency of breathing at the 5 mg/kg dose (Table 1) and a decrease in tidal volume at both doses (Table 2). The decrease in ventilation with the 5 mg/kg dose of Dex was due predominantly to an increase in inspiratory time at all time points (data not shown). Expiratory time increased significantly only at 15 min relative to saline (0.26 ± 0.01 vs. 0.31 ± 0.01 s; P < 0.01). Moreover, frequency tended to decrease with time (F = 15.2; P < 0.0001). This trend was noted with all doses of Dex. In contrast, neither tidal volume nor ventilation exhibited this time-related decrease.

Table 1. Breathing frequency of male and female rats treated with dextromethorphan Time, min 0 mg/kg 5 mg/kg 10 mg/kg 20 mg/kg Males 15 131 ± 6  127 ± 6  129 ± 5  117 ± 4  30 127 ± 7  118 ± 5  125 ± 5  113 ± 4  45 119 ± 8  114 ± 4  122 ± 4  113 ± 6  60 123 ± 7  114 ± 4  129 ± 5  104 ± 3* Females 15 132 ± 6  107 ± 4* 129 ± 9  132 ± 9  30 123 ± 6  104 ± 4* 115 ± 8  119 ± 8  45 108 ± 4  91 ± 3* 107 ± 6  111 ± 5  60 103 ± 4  91 ± 3  101 ± 6  103 ± 8 Values are means ± SE in breaths/min. * Significant difference relative to corresponding saline value at that time point, P < 0.001.

Table 2. Tidal volume of male and female rats treated with dextromethorphan Time, min 0 mg/kg 5 mg/kg 10 mg/kg 20 mg/kg Males 15 0.19 ± 0.02  0.19 ± 0.01  0.20 ± 0.01  0.20 ± 0.02  30 0.17 ± 0.01  0.17 ± 0.01  0.19 ± 0.02  0.19 ± 0.01  45 0.17 ± 0.01  0.17 ± 0.02  0.17 ± 0.01  0.19 ± 0.02  60 0.19 ± 0.02  0.16 ± 0.01  0.17 ± 0.01  0.18 ± 0.02  Females 15 0.24 ± 0.03  0.20 ± 0.02 0.21 ± 0.01 0.22 ± 0.03  30 0.25 ± 0.02  0.19 ± 0.02* 0.20 ± 0.02 0.25 ± 0.03  45 0.24 ± 0.04  0.20 ± 0.03  0.22 ± 0.03  0.24 ± 0.02  60 0.25 ± 0.03  0.23 ± 0.03  0.23 ± 0.03  0.26 ± 0.02 Values are means ± SE in ml/g × 100. Significant difference relative to corresponding saline value at that time point: * P < 0.001; P = 0.022.

In male rats, Dex had no effect on ventilation while they were breathing air (Fig. 2). With the exception of the 20 mg/kg dose of Dex at 60 min, neither frequency of breathing nor tidal volume was affected. Moreover, unlike female rats, male rats exhibited no time-dependent decrease in frequency of breathing.

Significant interactions between gender and treatment were noted for ventilation (F = 6.40; P < 0.0003), frequency of breathing (F = 5.51; P = 0.0011), inspiratory time (F = 11.76; P < 0.0001), and expiratory time (F = 4.30; P = 0.0051). A significant interaction between gender and time was only noted for frequency of breathing (F = 4.63; P = 0.011). No three-way interactions among gender, treatment, and time were observed with any parameter.

Female rats exposed to hypercapnia exhibited a smaller increase in ventilation relative to preceding air values when treated with 5 or 10 mg/kg of Dex relative to saline (Fig. 3). In contrast, male rats showed an increase in ventilation relative to saline with the 20 mg/kg dose of Dex. There were no significant interactions between gender and treatment, but each effect alone was significant (gender: F = 6.68; P < 0.01; treatment: F = 12.3, P < 0.0001). Baseline values before the hypercapnic challenge (Fig. 4) showed no effect of Dex in either gender. Relative to saline, Dex treatment did not significantly affect the ventilatory responses of male or female rats to hypoxia (F = 0.48; P = 0.694), but there was a gender effect (F = 5.23; P = 0.0245). Thus the response of male rats overall to hypoxia was greater than that of female rats.

Oxygen consumption was significantly depressed at 20 mg/kg of Dex in female rats at all time points (Fig. 5) and in male rats (Fig. 6) 45 and 60 min after injection. The times and doses at which ventilation and oxygen consumption were depressed did not overlap in female rats or correspond to the changes in ventilation in response to either hypoxia or hypercapnia.

In the second experiment, 10 mg/kg of Dex/Sal relative to Sal/Sal did depress ventilation in female rats (10.56 ml/min for Dex/Sal, P < 0.01; 4.61 ml/min for Sal/Sal, not significant). No such effect of Dex was noted in male rats. The interaction of aspartic acid and Dex is presented in Fig. 7. In male but not in female rats, aspartic acid (Sal/Asp) caused a significant depression in ventilation compared with 15- to 45-min data relative to Sal/Sal. With Dex pretreatment (Dex/Asp), this depression was no longer noted.

DISCUSSION

In the present study, Dex had disparate effects on ventilation in air and in response to hypercapnia in female compared with male rats. With 5 and 10 mg/kg of Dex, ventilation was depressed relative to saline in female rats, whereas these doses did not affect ventilation in male rats. The effects of Dex in female rats were due to an increase in inspiratory time that led to a decrease in frequency of breathing and a decrease in tidal volume at the 5 mg/kg dose. Tidal volume also showed a trend to decrease (P = 0.022) at 15 min with the 5 mg/kg dose and at 15 and 30 min with the 10 mg/kg dose. The decrease in ventilation was not due to a concurrent decrease in oxygen consumption. At the highest dose of Dex, oxygen consumption was depressed in both male and female rats. The relative selectivity of Dex on chemoreception was noted in that only hypercapnic but not hypoxic responses were affected. Moreover, a gender-specific response of Dex to hypercapnia was noted.

This is the first study designed to study the effects of Dex, a noncompetitive NMDA-receptor antagonist, on ventilation and oxygen consumption in male and female rats. Aside from a severe overdose, which has been shown to cause profound respiratory depression and even death, other anecdotal accounts of the effects of Dex on ventilation have included tachypnea in male rabbits and human subjects given high doses intravenously (3, 26). Dex is highly lipophilic and quickly enters the brain (29). There it acts on receptors in both the hypothalamus and brain stem regions, including the nucleus of the solitary tract, the nucleus ambiguus, hypoglossal nuclei, and pontine nuclei (6). These regions have been associated with the control of ventilation and energy expenditure (4, 18). Wu et al. (29) determined that 30 mg/kg of Dex administered subcutaneously into male Sprague-Dawley rats have a plasma half-life of 108 min, with an extremely high correlation between brain and plasma levels. There is some indication that the metabolism of Dex administered orally into male and female Sprague-Dawley rats is different. Ramachander et al. (20) reported that an oral dose of 10 mg/kg of Dex resulted in the plasma appearance of dextrorphan, a metabolite of Dex, within 5 min after administration in male rats, with peak values being reached by 15 min. In contrast, female rats exhibited higher concentrations for longer times. The authors attributed differences in the metabolism of Dex to sexual differences in Dex metabolism. In the present study, the effects of Dex on ventilation and oxygen consumption appeared rapidly in female rats, were markedly dissimilar at lower doses relative to male rats, and returned to baseline values, with the higher dose suggesting that metabolism of Dex itself was not sufficient to explain the differences noted. Moreover, the fact that Dex depressed ventilation in air and the ventilatory response to hypercapnia at the lower doses but not at the highest dose of Dex was unexpected but may possibly be associated with the effects of Dex on calcium and sodium channels or on other neurotransmitter systems such as dopamine (8, 15, 17).

Although the role of gender in the control of ventilation has been demonstrated (1, 27), the role of gender in NMDA-receptor-mediated control of breathing has not been studied. Its potential significance could be deduced from previous studies with aspartic acid (21, 23). The fact that aspartic acid caused a depression of breathing in castrated male rats equivalent to that seen in intact animals and ovariectomy had no effect in female rats suggests that the hormonal environment itself was not the only factor associated with the action of aspartic acid (21, 23). Moreover, by treating neonatal male rats with estradiol benzoate and female rats with testosterone propionate, the "gender-specific" ventilatory responses to aspartic acid could be reversed (23, 24). These results would suggest that factors organizing brain development in a sexual dimorphic manner during the early perinatal stage may influence the activity of aspartic acid actions on the control of breathing and possibly NMDA-receptor function. Repeating the present studies in female rats treated with testosterone propionate and male rats with estradiol benzoate and castrating adult animals may give insight into these possibilities.

Gender-related modulation of NMDA-receptor function has been studied in relation to MK-801 neurotoxicity, morphine and stress-induced analgesia, swim stress and ataxia, and hyperlocomotion (2, 10, 14, 16). In a recent review, Ellison (10) cited studies that showed the greater susceptibility of female rats to neural cortical vacuolization produced by MK-801 compared with male rats. Moreover, treatment of female rats with testosterone decreased this effect, whereas castration of male rats increased this effect, suggesting that hormonal manipulations could modulate NMDA-receptor function. Another example of gender differences in NMDA-receptor function was noted by Akinci and Johnston (2), who demonstrated that swim stress induced changes in MK-801 forebrain-receptor binding characteristics differently in male and female mice. Acute swim stress increased MK-801 binding in males but not in females. In a study by Hönack and Löscher (14), a low dose of MK-801 induced profound effects such as ataxia, hyperlocomotion, and head weaving in female but not in male rats. A threefold increase in dose resulted in similar behaviors in male rats. The investigators noted that these effects occurred rapidly, suggesting that they were not due to differential gender effects of MK-801 metabolism. Moreover, estrous-cycle state apparently did not alter the effectiveness of MK-801 in female rats. Thus genetics, gender, and, potentially, hormonal status (estrogen vs. testosterone) may influence NMDA-receptor modulation of ventilation and other physiological functions.

The marked effect of Dex on oxygen consumption has not been previously reported, although there is evidence that Dex can preserve ATP levels in the brain and reduce the production of lactate during brain ischemia (13). A decrease in oxygen consumption may contribute to these observations. In this study, the decrease in oxygen consumption was not accompanied by a decrease in ventilation, suggesting that Dex can cause a dissociation of the two parameters. This may be especially important when Dex is used as a therapeutic agent in such conditions as stroke and seizures (2, 28). A potential mechanism by which Dex may affect oxygen consumption could possibly be through its actions as a calcium-channel blocker (17). Although the present study did not investigate the effects of a channel-blocking agent on oxygen consumption in rats, administration of diltiazem, a calcium-channel antagonist, in hamsters has been shown to depress their oxygen consumption up to 75% of saline values (25). In that study, diltiazem also caused a depression of ventilation but at lower doses. Whether a portion of the effects of Dex noted in this study is due to its different effects on NMDA receptors, other neurotransmitter systems, calcium channels, and/or even, potentially, sodium channels in the brains of male and female rats needs to be determined.

In conclusion, this study demonstrated that Dex can affect ventilation of male and female rats breathing air or in response to hypercapnia differently. These effects were not due to a concurrent change in oxygen consumption. Moreover, pretreatment of male rats with Dex was able to prevent the aspartic acid-induced depression of ventilation in male rats. Finally, Dex may be a useful agent to explore NMDA-receptor modulation of ventilation in awake animals without many of the profound side effects such as seizure activity, hyperthermia, and excessive salivation reported in studies with MK-801.

ACKNOWLEDGEMENTS

I thank Darlene Hopkins-Rivera for technical help.

FOOTNOTES

Address reprint requests to E. H. Schlenker.

Received 25 January 1996; accepted in final form 20 June 1996.

REFERENCES