INTRODUCTION

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Nitric oxide (NO) has been shown to be involved in the control of breathing. Hypoxia activates the NO pathway in the central nervous system, contributing both to the induction and maintenance of the hypoxic ventilatory response (HVR) (1, 2). In addition, NO attenuates peripheral chemosensory discharge (3, 4). In conscious rats, NO synthase (NOS) inhibition increased the early (1-min) ventilatory response to hypoxia while reducing the late (30-min) ventilatory response (5).

NO has also been shown to be a pivotal mediator in the development of hypoxia-induced hypothermia. It is well known that hypoxia reduces body temperature (Tb) in neonates and small mammals (6). The systemic administration of NOS inhibitors has been shown to attenuate the decreases in both Tb and metabolic rate during hypoxia in awake rats (7, 8).

Obesity is an important risk factor for predicting respiratory disturbances, including alveolar hypoventilation and respiratory failure. The underlying mechanisms responsible for the depressed ventilation in obesity hypoventilation syndrome (OHS) have been unknown. The genetically obese Zucker (Z) rat exhibits many of the same respiratory deficits as obese humans, including reduced lung volumes, reduced chest wall compliance, and a blunted respiratory drive (9). The obese Z rat also displays a number of metabolic and thermoregulatory deficits, including a lower Tb (10) and blunted thermogenic response to cold exposure (13, 14). Furthermore, obese Z rats display decreased NOS activity in the hypothalamus, which may contribute to the hyperphagia and obesity (15). It is unknown, however, whether NO modulates ventilatory and metabolic regulation in response to hypoxia in obese Z rats. The purpose of the present study was to determine the role of NO in control of breathing and in thermoregulation in obese Z rats.

We hypothesized that the impaired thermogenic responses and the blunted respiratory drive previously observed in obese Z rats are secondary to altered NO levels in the brain. To test our hypothesis, we measured Tb, metabolic rates, and ventilatory responses to sustained hypoxia after the systemic administration of either N^G-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, or 7-nitroindazole (7-NI), a selective neuronal NOS inhibitor, in obese Z rats. A parallel study design was used with age-matched lean Z rats serving as nonobese controls. We studied relatively young Z rats to exclude any possible effects of chest wall limitation associated with obesity on ventilation (16).

	METHODS

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Animals

The studies were performed on male Zucker rats. Eight pairs of the lean phenotype (Fa/?) and obese phenotype (fa/fa) were purchased at 4 wk of age from Vassar College, Poughkeepsie, NY. The animals were 6 wk of age at the start of the study. One lean and one obese rat were housed per cage. Ambient temperature was maintained at 24° C with a 12-h light/dark cycle. All animals were provided with standard laboratory chow (Ralston Purina, St. Louis, MO) and water ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University at Buffalo.

Techniques and Measurements

Ventilation was measured by the barometric technique of plethysmography. A cylindrical Plexiglas chamber with a volume of 4 liters was used for the measurements of ventilation. The rat was placed in the chamber within a restrainer, which did not permit backward rotation. The animal chamber had an inlet tube that was connected to pressurized air tanks (BOC Gases). Inlet flow was regulated at 2 L/ min by a flowmeter (Dwyer Instruments Inc., Michigan City, IN). The concentrations of inflowing or outflowing O₂ and CO₂ were monitored by an Ametek S-3A/I O₂ analyzer and an Ametek CD-3A CO₂ analyzer. The calibration of the gas analyzers were checked once daily, using certified calibration gases (BOC gases). To measure ventilation, the chamber was completely sealed after momentarily interrupting the flow through it, and the oscillations in pressure caused by breathing were recorded by a sensitive pressure transducer (Statham Laboratories, Oxnard, CA). The signal was received, amplified (Grass Instruments Co., Quincy, MA) and displayed on an oscillographic strip chart recorder (Grass Polygraph). An average of 50 breaths was recorded on paper at a speed of 10 mm/s. Injection and withdrawal of 0.3 ml of air with a 1-ml syringe were performed several times during the recording for calibration purposes. Body temperature was measured continuously by a thermometer probe (Yellow Springs Instruments, Yellow Springs, OH) placed at least 5 cm into the rectum. The thermometer was calibrated with a mercury thermometer within a temperature range of 35 to 40° C. Chamber temperature and relative humidity were monitored by a flow-through probe (Fisher Scientific, Pittsburgh, PA) mounted within the chamber. The temperature inside the chamber was controlled at 26-27° C by heating or cooling. Barometric pressure values to the nearest hour were obtained from the Internet posting of the US weather bureau located at the Buffalo International Airport.

Respiratory frequency (f) was calculated directly from the ventilation-induced pressure swings. Tidal volume (VT) was obtained as a function of the pressure change inside the chamber. Pulmonary ventilation (E) was calculated (E = VT × f) and expressed in either raw data (ml/min) or per body weight (BW) (ml/min/kg). Oxygen consumption (O₂) was calculated from the inflow-outflow O₂ differences multiplied by the gas flow, neglecting the small error introduced by a respiratory quotient less than unity. All O₂ values are presented at Standard Temperature and Pressure Dry (STPD), and expressed in either raw data (ml/min) or per unit of effective body mass (EBM) because lean and obese rats of the same size have different body compositions (17). EBM for lean and obese Z rats was calculated as 1.00 × M^0.75 and 0.86 × M^0.75, respectively, where M is the body weight (kg) of the animal.

Experimental Protocol

In order to reduce the stress level during the experiment, all animals were habituated to an intraperitoneal injection of 0.4 ml of saline, the insertion of the thermoprobe, and the restraining device within the chamber for 60 min on two successive days prior to the first experimental study. Each animal was repeatedly tested at 3-day intervals after an intraperitoneal injection of equal volumes of either 1.67 ml/kg of vehicle (dimethyl sulfoxide: DMSO), 100 mg/kg of L-NAME, or 25 mg/kg of 7-NI. Selected dosages for both L-NAME (5, 18) and 7-NI (8, 19) to inhibit NOS have been previously validated for the rat. The solutions were prepared daily (dose: L-NAME, 60 mg/ml; 7-NI, 15 mg/ml) and placed in vials labeled as blinded-solutions I, II, or III. To reduce the effects of adaptation of animals to repeated tests, the agents were given in a randomized design on Days 1, 4, and 7. The investigator involved in the actual testing remained blinded to the contents of the vials until the whole study was completed and analyzed. To minimize any potential differences caused by circadian rhythms, experiments were begun in each animal at the exact same time between 8:00 A.M. and 3:00 P.M.

After the administration of the agent and the insertion of the rectal thermoprobe, the rat was placed into the barometric chamber within the restrainer, and it breathed room air for 30 min followed by 30 min of hypoxia (10% O₂, balance N₂). Thereafter, animals were exposed to room air for 10 min. Ventilatory data were collected at 20 and 30 min postdrug administration during room air, at 33, 40, 50, and 60 min postdrug administration during hypoxia, and at 65 and 70 min postdrug administration during room air. Metabolic variables were recorded at 10-min intervals.

Statistical Analysis

When comparing the differences between lean and obese Z rats, all data were expressed in absolute values. The differences between two groups were analyzed by one-way analysis of variance (ANOVA). To compare the differences among the responses after vehicle, L-NAME and 7-NI administration within a group, all data were expressed in corrected values for body weight (BW) because each animal was repeatedly tested at 3-d intervals and its BW changed at each experimental date. The differences between three agents were analyzed by two-way ANOVA with repeated measurements. When significance was indicated, a post-hoc t test with Bonferroni's correction for multiple comparisons was used for point-by-point differences. In all cases, a p value < 0.05 was considered statistically significant. All data presented in the text, tables, and figures represent means ± SEM.

	RESULTS

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The obese Z rats weighed about 20% more than the age-matched lean animals (156 ± 11 versus 130 ± 7 g, p < 0.05; unpaired t test).

Comparison between Lean and Obese Rats with Vehicle Injection

The changes in Tb, O₂, E, f, and VT during room air and 10% O₂ exposure for 30 min in lean and obese rats with vehicle injection are shown in Figure 1. Tb in obese Z rats during room air was significantly lower than that of the lean Z rats (37.7 ± 0.1 versus 38.1 ± 0.1° C, p < 0.01)(Table 1 and Figure 1). Hypoxia induced a more pronounced drop in Tb and O₂ in lean rats than obese rats (Figure 1). The decreases in Tb (hypoxic hypothermia: Tb) and O₂ (hypoxic hypometabolism: O₂) after 30 min of hypoxia from the baseline values were significantly smaller in obese rats than in lean rats (Tb: 0.3 ± 0.1 versus 0.8 ± 0.1° C, p < 0.01, O₂: 0.67 ± 0.05 versus 1.00 ± 0.06 ml/min, p < 0.01, respectively) (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Comparisons of body temperature (Tb), oxygen consumption ( O2), ventilation ( E), respiratory frequency (f), and tidal volume (VT) between lean (open circles) and obese (closed squares) Z rats with vehicle injection during room air and hypoxia (10% O2). Recording of data started 20 min after injection of vehicle. *p < 0.05, **p < 0.01. Significantly different in the values at the same time point between lean and obese rats. Values represent mean ± SEM.

View larger version (26K): [in this window] [in a new window]   Figure 2.   The decrease in Tb (hypoxic hypothermia: Tb) and O2 (hypoxic hypometabolism: O2) after 30 min of hypoxia from the base line values in lean and obese rats with vehicle injection. **p < 0.01. Significantly different from the mean value of lean rats. Values represent mean ± SEM.

During room air, obese rats breathed with a significant higher f and a lower VT than lean rats, resulting in the similar E value compared with lean rats (Table 1). Lean rats showed an abrupt increase in E 3 min after hypoxia exposure followed by a significant decrease at 20 and 30 min of hypoxia compared with the response at 3 min of hypoxia (biphasic pattern). This biphasic pattern was mainly due to a reduction in f (Figure 1). In contrast, obese rats displayed an initial increase inE that plateaued throughout the 30 min of hypoxia exposure (Figure 1).

Effects of L-NAME and 7-NI during Room Air

In lean rats, Tb dropped significantly by ~ 0.2° C after L-NAME and, more markedly, by ~ 1.1° C after 7-NI compared with control (p < 0.05, p < 0.01, respectively). Similarly, O₂ /EBM was significantly less in lean rats with L-NAME and 7-NI, than in control (p < 0.05, p < 0.01, respectively) (Table 1 and Figure 3). E/BW of lean rats tended to be reduced with L-NAME and was reduced significantly with 7-NI, compared with control. The decreases in E/BW were due to marked reduction in f (Table 1). E/O₂ was not affected by either agent in lean rats (Table 1).

View larger version (34K): [in this window] [in a new window]   Figure 3.   Effects of vehicle control (open circles), L-NAME (closed triangles) or 7-NI (closed circles) injection on body temperature (Tb), oxygen consumption ( O2/EBM), ventilation ( E/BW) and ventilatory equivalent for O2 ( E/ O2) during room air and hypoxia (10% O2) in lean and obese Z rats. Recording of data started 20 min after injection of an agent. *p < 0.05, **p < 0.01. Significantly different from the value of control at the same time point. Values represent mean ± SEM.

In marked contrast, obese rats exhibited no change in Tb, O₂/EBM, E/BW, f, VT/BW and E/O₂ (ventilatory equivalent for O₂) during room air breathing after either L-NAME or 7-NI administration (Table 1 and Figure 3).

Effects of L-NAME and 7-NI during Hypoxia

The time-dependent changes in Tb, O₂/EBM, E/BW, and E/O₂ during 30 min of hypoxia in lean and obese rats are shown in Figure 3, and the decreases in Tb (Tb) and O₂/ EBM (O₂/EBM) from the base line after 30 min of hypoxia are shown in Figure 4. In lean rats, the change in Tb during hypoxia was not altered after L-NAME, but significantly altered after 7-NI administration compared with control rats. The change in O₂/EBM during hypoxia was significantly affected with both L-NAME and 7-NI compared with control animals. However, in obese rats, there were no significant differences in Tb and O₂/EBM during hypoxia between three agents (Figure 4).

View larger version (34K): [in this window] [in a new window]   Figure 4.   Effects of L-NAME and 7-NI on hypoxic hypothermia (Tb) and hypoxic hypometabolism ( O2/EBM) in lean and obese Z rats. In lean rats, L-NAME and 7-NI significantly decreased the magnitude of Tb or O2/EBM. However, in obese rats, there were no significant differences in Tb and O2/EBM among three agents. *p < 0.05, **p < 0.01. Significantly different from the value of control. Values represent mean ± SEM.

In lean rats, E/BW was significantly reduced with L-NAME at 3 and 10 min after hypoxia exposure and more markedly decreased with 7-NI at all points during hypoxia, compared with control. On the other hand, E/BW in obese rats was unaffected by the administration of either L-NAME or 7-NI (Figure 3).

E/O₂ in lean rats was significantly reduced with 7-NI during hypoxia, but not with L-NAME compared with control. In obese rats, neither L-NAME nor 7-NI affected E/O₂ during 30 min of hypoxia (Figure 3).

	DISCUSSION

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The important results of the present study are that: (1) the patterns of Tb, O₂ and E responses to sustained hypoxia were different between lean and obese rats; (2) Tb and O₂, in lean rats decreased significantly with NO antagonists compared with control during room air, whereas they were unaffected in obese rats; (3) NO antagonists attenuated the hypoxia-induced hypothermia or hypometabolism only in lean rats, but not in obese rats; and (4) NO antagonists induced marked decreases in E during room air and hypoxia in lean rats, whereas E was virtually unaffected by either agent in obese rats. The present results indicate that NO plays little role in thermoregulation and control of breathing in obese Z rats, and they suggest that the blunted thermoregulatory and ventilatory responses to hypoxia in obese Z rats may be the results of reduced activity of NOS in the central nervous system.

Effects of NOS Inhibition on Thermoregulation

The present results in lean Z rats during room air agree with recent reports in Wistar rats showing a decrease in Tb after intravenous injection of L-NAME (7) or intraperitoneal injection of 7-NI (8). Although it has been suggested that NO is required for the production of fever because NOS inhibition reduced the febrile response to lipopolysaccharide (LPS) (20), the exact mechanism underlying the reduction in Tb by NO inhibition is unknown. It has been demonstrated that L-NAME elicits hypothermia by reducing the firing rate of sympathetic nerves innervating intrascapular brown adipose tissue (BAT) which contributes to heat production (21). However, L-NAME should also potentially reduce heat loss because it induces vasoconstriction in peripheral vessels of the skin.

At the selected dose (100 mg/kg), L-NAME decreases not only endothelial NO but also neuronal NO production in the rat (18). The present responses of Tb and O₂ in lean rats suggest that L-NAME might cause a more pronounced decrease in heat production than a reduction in heat loss. On the other hand, 7-NI is believed to elicit hypothermia by directly decreasing NO in the thermoregulatory centers that ultimately control heat production in BAT (8).

In response to hypoxia exposure, we observed that hypoxia-induced hypothermia or hypometabolism in lean rats was inhibited both by L-NAME and 7-NI (Figure 4). Our findings are consistent with those of Branco and colleagues (7) in which the hypoxic-hypothermic response could be attenuated by intravenous (30 mg/kg) or by intracerebroventricular (1 mg/kg) administration of L-NAME. Furthermore, Gautier and Murariu (8) showed that 7-NI reduced hypoxia-induced hypometabolism, but that the hypothermia persisted. Although it has been speculated that NO in the central nervous system may wholly or partly mediate hypothermia or hypometabolism in hypoxia (7, 8), the precise mechanism is still unknown.

In sharp contrast to the results in lean rats, L-NAME and 7-NI administration failed to affect Tb and O₂ in obese rats, not only during normoxia but also during hypoxia. Some metabolic abnormalities have been noted previously in obese Z rats, including decreased BAT (22) and increased white adipocyte cell size (23). Many of these anomalies are under the control of the sympathetic nervous system, which exhibits reduced activity in obese rats (24, 25). Furthermore, the obese Z rat has been reported to exhibit a number of thermoregulatory deficits when compared with lean littermates. These deficits include a lower body temperature during normoxia (10), reduced thermogenic responses to cold exposure (13, 14), to saline-injection stress, and to LPS- induced fever (26), and a reduced circadian rhythmicity (27). In addition to the various anomalies previously reported for the obese Z rat, we have made a new observation that the hypoxia-induced hypothermic or hypometabolic response is blunted in obese Z rats when compared with lean Z rats.

With regard to neuromodulation in the hypothalamus, it has been reported that NOS activity is decreased in obese rats when compared with lean rats (15), suggesting that the genetic defect responsible for the hyperphagia of the obese Z rat involves an uncoupling of NO-cyclic GMP activity. We therefore suggest that the unchanged Tb and O₂ after administration of NO antagonists in obese rats may be due to a primary or basal reduction in NOS activity in the thermoregulatory centers. In addition, reduced NO levels in the central nervous system may explain why the set-point for Tb is lower in obese rats than in lean rats (10).

Effects of NOS Inhibition on Ventilation

Room air. It is now well established that NO exerts a role in respiratory control by enhancing the excitability of the neurons involved in the generation of central respiratory activity (1, 2, 28). In consequence, a reduction of ventilation would be expected after administration of NO antagonists. In the present study, we noted a marked decrease in E after L-NAME or 7-NI injection compared with control in lean Z rats, whereas no change was observed in obese rats. However, when the concomitant decrease in O₂ was taken into account, the ventilatory equivalent for O₂ (E/O₂) was not changed with either agent in lean rats. Our findings in lean rats confirm the report of Gautier and Murariu (8), which showed that intraperitoneal injection of 7-NI caused a significant reduction inE with a slight increase in E/O₂ in awake Wistar rats. We conclude that, in normoxia, the reduction in ventilation after NO antagonist is mainly due to its hypothermic or hypometabolic action.

Hypoxia. Respiratory drive during hypoxia is determined by a balance between the stimulation of the peripheral chemoreceptors and the central depression of hypoxia on respiration. It has been postulated that the early component of HVR is mainly due to the activation of the peripheral chemoreceptors, whereas the later response is modulated by a variety of neurotransmitters and factors associated with hypoxia (29). With regard to NO as a neurotransmitter, NO attenuates the peripheral chemosensory discharge (3, 4), whereas, by contrast, NO derived from central neuronal NOS enhances phrenic nerve output during hypoxia (1, 2). Hence, the same molecule subserves both inhibitory and excitatory functions at different sites of the hypoxic chemotransduction pathway. Gozal and colleagues (5) confirmed this notion as they observed that L-NAME increased the early (1 min) ventilatory response to hypoxia, whereas it reduced the late (30-min) ventilatory response in awake Sprague-Dawley rats. In contrast, we found a marked decrease in ventilation at 3 min of hypoxia in L-NAME treated lean rats. The carotid sinus nerve afferent input projects to the glutamatergic synapses in the nucleus tractus solitarii (NTS) where NO acts as a retrograde messenger contributing to the augmentation of ventilation during hypoxia (2). It is, therefore, possible that L-NAME may block not only endothelial NOS in the carotid body, but also neuronal NOS in the NTS simultaneously. Consequently, the net E response after L-NAME administration may consist of both increased and decreased components of ventilation. Therefore, in Sprague-Dawley versus lean Z rats, the effect of NO inhibition on the early response seems to depend on the relative importance of the action of NO in either the peripheral or the central pathway. We suggest that the major site of action of NO during hypoxia must be central in lean Z rats because 7-NI, which inhibits NOS solely in the brain in vivo (30), attenuated both the early and the late E response to hypoxia. Even though a concomitant decrease in O₂ during hypoxia was considered, E/O₂ was significantly reduced after 7-NI administration compared with control. Our results with 7-NI confirm those of Gozal and colleagues (5) showing that neuronal NOS inhibition by SMTC induced marked reductions in the late (30-min) ventilatory response to hypoxia in awake rats. Most recently, Gautier and Murariu (8) have also shown that 7-NI decreased the E or E/O₂ response to 15 min of hypoxia in awake Wistar rats.

In obese Z rats, NO probably plays little role in control of breathing because neither L-NAME nor 7-NI had any effects on resting ventilation and HVR. We therefore speculate that the lack of response to NO antagonists on ventilation in obese Z rats may be due to an absent or reduced activity of neuronal NOS in the central respiratory control centers. Indeed, NOS activity has been shown to be reduced in the hypothalamus of obese Z rats when compared with lean rats (15).

Interaction between Thermoregulation and Ventilation

The lean Z rats exhibited an abrupt increase of E in response to hypoxia followed by a gradual decline with sustained hypoxia (biphasic pattern). In contrast, obese Z rats displayed an initial increase in E that plateaued with sustained hypoxia exposure (Figure 1). Our observation represents the first description of the time course for the ventilatory response to sustained hypoxia in Z rats. The ventilatory response to sustained hypoxia is modified by many factors, including state of consciousness, PCO₂ level, metabolic rate, neurotransmitters in the central nervous system, and neurochemical modulation of peripheral chemoreceptor function (29). In neonates and small mammals, hypoxic hypoventilation is often associated with hypoxic hypothermia or hypometabolism (6). Moreover, it has been demonstrated that combined hypothermia and hypoxia interact to cause a depression in ventilation in awake rats (31). In the present study, the depression in E during sustained hypoxia in lean rats was mainly due to a decrease in f, whereas VT remained unaltered. In addition, 7-NI completely abolished the biphasic pattern of HVR in lean rats (Figure 3) while it simultaneously attenuated the hypoxia-induced hypothermia and hypometabolism (Figure 4). Consequently, the patterns of Tb, O₂, and E responses after 7-NI administration in lean rats were very similar to those of obese rats. Therefore, a possible mechanism for the biphasic pattern of HVR in lean rats may be an appropriate decrease in ventilation associated with the hypoxic hypometabolism. Furthermore, we suggest that brain NO may modulate both ventilatory and thermal responses to hypoxia as a common mediator in lean rats.

With respect to the central site of action of NO in the rat, the posterior hypothalamic area (PHA) has been shown to play a pivotal role. Maskrey and Hinrichsen (32) showed that the PHA is a likely site for the interactions between hypoxia and hypothermia. Most recently, Gautier and Murariu (8) also suggested that both the hypometabolic and the ventilatory responses to hypoxia must share brain NO as a common mediator. Taken together, hypoxia-induced hypothermia and hypoventilation may, at least in part, be mediated by NO in the brain. In obese Z rats, the small drop in Tb or O₂ and the lack of biphasic decrease in E during hypoxia is probably related to a common dysfunction of thermoregulation and respiratory regulation in response to hypoxia via altered NO mechanism.

On the other hand, the obese Z (fa/fa) rats have a defect in leptin access to the brain because of a leptin receptor mutation, which may account for the development of their obesity (33). Calapai and colleagues (34) recently demonstrated in the mouse that the declines in food intake and body weight gain after leptin administration were antagonized by L-arginine (NO substrate) administration, and diencephalic NOS activity was significantly reduced after intravenous leptin injection, suggesting that the brain L-arginine/NO pathway might be involved in the central effect of leptin. Furthermore, NOS activity in the hypothalamus has been shown to be reduced in obese Z rats when compared with lean Z rats (15). Thus, it may be interpreted that disrupted leptin transport into the brain may contribute to the NO dysfunction in thermoregulation and respiratory control observed in obese Z rats. If this is the case, the question can be raised whether high fat diet-induced obesity in lean Z (Fa/?) rats would also display the same blunted responses in Tb and E to NOS inhibition as obtained from weight-matched obese Z (fa/fa) rats. Elevated plasma levels of leptin have been reported in obese humans (35), wild-type mice (36), and lean Z (Fa/Fa) rats with diet- induced obesity (33). Leptin suppresses NOS activity in the brain (34) but has a direct stimulating effect on respiratory control centers (36). Thus, it is likely that increased plasma leptin levels induced by a high fat diet may cause a dysfunction in the brain L-arginine/NO pathway, although leptin may potentially prevent respiratory depression in obesity. We therefore speculate that not only genetic obesity but also dietary obesity may well have an abnormal NO mechanism in thermoregulation and control of breathing. Further studies, however, will be necessary to answer this question.

In summary, in obese Z rats, NO probably plays little role in thermoregulation and control of breathing because neither L-NAME nor 7-NI had any effects on temperature, metabolism, and ventilation during room air and hypoxia. We therefore suggest that the blunted thermoregulatory and ventilatory response to hypoxia in obese Z rats may be attributed to a deficiency in brain NO. Although a role of NO in OHS has not been previously proposed, the current results suggest that the reduced NOS activity in the brain may, in a part, contribute to the pathogenesis of OHS in humans.