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Am J Physiol Regul Integr Comp Physiol 293: R1350-R1356, 2007. First published July 11, 2007; doi:10.1152/ajpregu.00318.2007
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ENVIRONMENTAL, EXERCISE AND RESPIRATORY

Hypoxic ventilatory response during light and dark periods and the involvement of histamine H1 receptor in mice

Yasuyoshi Ohshima,1,2 Michiko Iwase,1 Masahiko Izumizaki,1 Takashi Ishiguro,1 Mitsuko Kanamaru,1 Hideaki Nakayama,2 Fumitake Gejyo,2 and Ikuo Homma1

12nd Department of Physiology, Showa University School of Medicine, Shinagawa-ku, Tokyo; and 2Division of Respiratory Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Submitted 7 May 2007 ; accepted in final form 11 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ventilation oscillates throughout a day in parallel with oscillations in metabolic rate. Histamine affects ventilation and the balance of the energy metabolism via H1 receptors in the brain. We tested the hypothesis that the ventilatory response to hypoxia varies between light and dark periods and that histamine H1 receptors are required for the circadian variation, using wild-type (WT) and histamine H1 receptor knockout (H1RKO) mice. Mice were exposed to hypoxic gas (7% O2 + 3% CO2 in N2) during light and dark periods. Ventilation initially increased and then declined. In WT mice, minute ventilation (VE) during hypoxia was higher in the dark period than in the light period, which was an upward shift along with the baseline ventilation. Hypoxia decreased the metabolic rate, whereas O2 consumption (VO2) and CO2 excretion were higher in the dark period than in the light period. However, in H1RKO mice, changes in VE during hypoxia between light and dark periods were minimal, because VE was increased relative to VO2, particularly in the light period. In H1RKO mice, the HCO3– concentration and base excess values were increased in arterial blood, and the level of ketone bodies was increased in the serum, indicating that metabolic acidosis occurred. Respiratory compensation takes part in the VE increase relative to VO2 during hypoxia. These results suggested that changes in VE during hypoxia vary between light and dark periods and that H1 receptors play a role in circadian variation in VE through control of the acid-base status and metabolism in mice.

H1 receptor knockout mice; hypoxia; ventilation; metabolism; circadian variation

VENTILATION, SIMILAR TO OTHER physiological and behavioral variables, endogenously oscillates throughout the day (12, 35, 38). This circadian rhythm of ventilation could be caused by neural inputs from the circadian pacemaker located in the suprachiasmatic nuclei or by indirect influences on ventilatory control via the circadian rhythms of other variables (38). In nocturnal animals, metabolism and body temperature increase in the dark period and decrease in the light period in parallel with similar changes in ventilation (12, 35, 39).

Circadian rhythm is modified by histaminergic neurons in the hypothalamus (37, 42). Histaminergic neurons are localized in the tuberomammillary nucleus and send axons to various areas, including the hypothalamic nuclei, the pons, and the medulla (10, 15, 44). Three histamine receptors (H1, H2, and H3 receptors) have so far been identified in the brain. Of these, histamine H1 receptors have crucial roles in the circadian rhythms of locomotor activity (11), feeding and drinking (14), and arousal reactions (23). Histamine H1 receptors are also involved in thermoregulation and the balance of the energy metabolism (8, 26, 34), both of which are closely related to ventilation and metabolic rate. Indeed, histamine contributes to ventilatory control during hyperthermia, hypercapnia, and hypoxia via H1 receptors (13, 17–19, 28). These findings show that histamine plays a role not only in circadian rhythm but also in ventilatory control and other autonomic functions.

The respiratory pattern is generated by the network system in the lower brain stem that is regulated by an autonomic metabolic control system via central and peripheral chemoreceptors. Hypoxia induces a biphasic ventilatory response composed of an initial increase induced by activation of arterial chemoreceptors, with a subsequent decline accompanied by a decrease in metabolic rate, the so-called hypoxic ventilatory decline (HVD) (2, 43). It has also been suggested that HVD might be mediated in part by time-dependent changes in the activity of the carotid body (4). HVD is mediated by inhibitory neurotransmitters such as GABA, adenosine, and endogenous opioids (30). Recently, histamine has been proposed to contribute to HVD via H1 receptors in given conditions in in vivo and in vitro states (5, 13, 17).

In an earlier study we showed circadian variation in ventilation and metabolism under normoxic conditions in mice (12). In humans, it has been observed that hypercapnic ventilatory control and end-tidal PCO2 vary with the light-dark cycle (36, 38). However, little is known about whether ventilation in response to acute hypoxia shows circadian variation. We hypothesized that the minute ventilation (VE) response to hypoxia varies between light and dark periods and that histamine H1 receptors are required for the circadian variation in the VE response. We tested these two hypotheses using histamine H1 receptor knockout (H1RKO) and wild-type (WT) mice.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. H1RKO mice (Kyushu University, Fukuoka, Japan) (11) were backcrossed to C57BL/6 mice at Showa University. H1RKO mice and WT mice were maintained under pathogen-free conditions as described previously (13, 28). Mice were provided with food and water ad libitum, housed at a controlled temperature (24°C), and exposed to a daily 12:12-h light-dark cycle (light off at 2000). Male mice ages 8–11 wk were used for experiments. All experiments were performed at an environmentally controlled temperature (24–25°C) in the light period (1000–1600) and in the dark period (2200–0400). The study protocol was approved by the Showa University Animal Experiments Committee.

Measurement of lung ventilation. Respiratory variables were measured using a whole body unrestrained plethysmograph (WBP) (PLY3211; Buxco Electronics, Sharon, CT). This system is described elsewhere in detail (20). In brief, the WBP chamber received air at a flow rate of 1 l/min through a flow pump-reservoir system (PLY1020; Buxco Electronics), and the differential pressure between the experimental and reference chambers was measured using a differential pressure transducer. The pressure signal was amplified and then integrated using data analysis software (Biosystem XA for Windows; Buxco Electronics). A calibration volume of 1 ml of ambient air was introduced into the chamber with a syringe. Room temperature, humidity, and barometric pressure were measured routinely before respiratory measurements, and these values were taken as the conditions in the chamber during respiratory measurements. Animal body temperature was estimated as 37°C based on mean rectal temperature in conscious mice, as described previously (28). The chamber was protected from visual stimulation by a screen. Respiratory frequency (fR, breaths/min), tidal volume (VT, ml BTPS; BTPS, body temperature and pressure, saturated with water vapor), and minute ventilation (VE, ml BTPS/min) were computed breath by breath throughout all baseline and experimental photoperiods. VT and VE were normalized per 10 g of body weight.

WT mice (n = 10) and H1RKO mice (n = 10) were used for measurement of ventilatory variables during normoxia and hypoxia in the light period. Another set of mice (each genotype, n = 10) was used in the dark period to avoid repeating measurements and aging effects. Each mouse was placed in a WBP chamber and acclimatized to the chamber for at least 90 min before measurements were taken. After acclimatization to the WBP, fR was ~250 breaths/min and VE was ~30 ml·10 g–1·min–1 during breathing of room air. In this condition, air was perfused for 30 min, and then hypoxic gas (7% O2 + 3% CO2 balanced in N2) was perfused for 20 min. Hypoxic gas contained 3% CO2 to compensate for the reduction in arterial PO2 caused by "blowing off" of CO2 during elevated ventilation associated with hypoxia (16, 33). Tests for hypoxic gas exposure were performed in light and dark periods.

Measurement of aerobic metabolism. Aerobic metabolism was measured using an open-circuit system with a magnetic-type mass spectrometer (ARCO-1000; ARCO System, Chiba, Japan). Gases were collected from the inflow and outflow of an animal chamber (120 x 70 x 60 mm), and the levels of oxygen (O2) and carbon dioxide (CO2) were analyzed. O2 consumption (VO2, ml STPD/min; STPD, standard temperature and pressure, dry), CO2 excretion (VCO2, ml STPD/min), and respiratory exchange ratio (R) were computed every minute. VO2 and VCO2 were normalized to kilograms of body weight.

WT mice (n = 10) and H1RKO mice (n = 10) that were not used for the measurement of lung ventilation were measured for aerobic metabolism during normoxia and hypoxia in the light period. Another set of mice (each genotype, n = 10) was used in the dark period. Each mouse was placed in the chamber and acclimatized to the chamber for at least 90 min. After acclimatization, the mouse was delivered air (30 min) and then hypoxic gas (7% O2 + 3% CO2 in N2, 20 min). VO2, VCO2, and R values in the stable state over a 10-min period during air gas inhalation were averaged; these were regarded as resting values during normoxia. Metabolic variables during hypoxia represent the average values during a stable 3-min period around 20 min after the commencement of hypoxic gas inhalation. The time course of the metabolic response to hypoxia was determined at 10, 15, and 20 min after inhalation of hypoxic gas commenced. The values for metabolism during a 3-min period around the objective time were averaged.

Analysis of arterial blood gases. Arterial blood gases were measured in just the light period in a different set of mice from those used in other experiments. To sample blood, an arterial catheter was inserted into the right carotid artery and ligated with a surgical thread under anesthesia (pentobarbital sodium, 25 mg/kg ip) as described previously (16). After surgery, each mouse was placed in a plastic chamber (90 x120 x 50 mm) in which it was allowed to recover from anesthesia for at least 4 h. Body temperature was maintained by a warming lamp.

WT and H1RKO mice were measured for blood gases. First, a mouse in the plastic chamber was delivered air for 20 min, and then arterial blood (120 µl) was collected into heparinized glass sampling tubes (MC0020; AVL Scientific, Roswell, GA); this was regarded as the normoxic condition (WT, n = 9; H1RKO, n = 9). The blood sample was analyzed immediately with a blood gas analyzer (OPTI CCA; AVL Scientific). The mouse was then delivered hypoxic gas for 20 min, and blood was again collected and analyzed; this was regarded as the hypoxic condition (WT, n = 7; H1RKO, n = 7). Arterial blood gases were analyzed for pH, arterial O2 partial pressure (PaO2), arterial CO2 partial pressure (PaCO2), concentrations of hydrogen carbonate ([HCO3–]), and base excess (BE).

Measurement of glucose and lactate levels in blood. A small droplet of whole blood was collected from the tail vein by a small cut made with a razor edge and was tested for glucose level using the FreeStyle blood glucose monitoring system (Kissei Pharmaceutical, Matsumoto, Japan). It was then tested for lactate level using Lactate Pro (Arkray, Kyoto, Japan). The levels of glucose and lactate in WT mice (n = 10, respectively) and H1RKO mice (n = 10, respectively) were measured in both photoperiods under ad libitum feeding conditions.

Measurement of glucose and lipid-related substances in serum. Glycated albumin and lipid-related substances in serum were examined in the light period (1300–1400; WT, n = 11; H1RKO, n = 11) and the dark period (2200–2300; WT, n = 11; H1RKO, n = 11). Under ad libitum feeding conditions, blood was collected from the right ventricle under ketamine anesthesia (50 mg/kg ip) and centrifuged to yield serum. Serum levels of substances were assayed using commercially available kits. Analyses were performed for total cholesterol (TC; Wako Pure Chem, Osaka, Japan), low-density lipoprotein cholesterol (LDL; Daiichi Pure Chem, Tokyo, Japan), high-density lipoprotein cholesterol (HDL; Daiichi Pure Chem), triglyceride (TG; Wako Pure Chem), free fatty acids (FFA; Eiken Chem, Tokyo, Japan), ketone bodies (Wako Pure Chem), and glycated albumin (Asahi Kasei Pharma, Tokyo, Japan).

Statistical analysis. Data are means ± SE. To assess the effects of hypoxia during the different photoperiods and in different genotypes, we performed three-way analysis of variance (ANOVA) for repeated measures for respiratory variables (fR, VT, and VE) and metabolic variables (VO2, VCO2, and R). Genotype and photoperiod factors were the between-group factors, whereas time course was the within-group factor used as a repeated measurement. When an overall significant main effect was found for photoperiod, post hoc analyses were performed using t-test with Bonferroni correction for multiple comparisons. Increased amplitudes of VE from the resting state were analyzed between light and dark periods using Student's t-test. The ratios of VE to VO2 in different periods and genotypes were also analyzed using Student's t-test, as were arterial blood gases (pH, PaCO2, PaO2, [HCO3–], and BE) and blood and serum variables. For these analyses, we used SPSS II software (SPSS Japan, Tokyo, Japan). Statistical significance was set at P < 0.05, and the P values were adjusted for multiple comparisons.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxic ventilatory response to hypoxia during the light and dark periods. Figure 1, A–F, shows ventilatory variables in response to hypoxia during light and dark periods. In WT mice, hypoxia increased fR, VT, and VE, all of which reached a peak 2 min after the start of inhalation and then declined. The levels of fR and VE in normoxia and hypoxia were higher in the dark period than in the light period, showing circadian variation. However, the amplitude of the initial increase, the VE difference from the resting state to the peak at 2 min, was not significantly different between photoperiods. This indicated that hypoxic response was shifted upward following a higher basal level of ventilation. In H1RKO mice, ventilatory variables in a course of hypoxia showed a profile similar to those in WT mice. Meanwhile, the circadian variations in fR and VE were small (Fig. 1, D–F).


Figure 1
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  		Fig. 1. Measurement of respiratory variables in response to hypoxic gas exposure (7% O2 + 3% CO2 in N2) in wild-type (WT) mice (A–C) and histamine H1 receptor knockout (H1RKO) mice (D–F). Open symbols indicate values during the light period, and filled symbols indicate values during the dark period. Changes in respiratory frequency (fR; A and D), tidal volume (VT; B and E), and minute ventilation (VE; C and F) are shown. A significant main effect was observed for time and photoperiod for fR, VT, and VE and for genotype for VT and VE (3-way ANOVA, P < 0.05). A significant effect of the photoperiod for ventilatory variables at the corresponding time was also observed (*P < 0.01, t-test with Bonferroni correction).

 
Statistical analysis supports a reduced circadian variation in ventilatory variables in H1RKO mice. A three-way ANOVA yielded significant main effects of time course and photoperiod for all variables (fR, VT, and VE, P < 0.05). A significant main effect of genotype and a significant interaction of genotype x time course were found for VT and VE (P < 0.05). A significant interaction of the genotype x photoperiod for fR (P < 0.05) was also observed. A post hoc test showed significant differences between light and dark periods in fR and VE in WT mice (P < 0.01, t-test with Bonferroni correction) but not in H1RKO mice. These analyses indicated that VE and its components, fR and VT, display circadian variation but that the hypoxic response shows an upward shift without any change in the profile. There were also genotype-specific differences in fR, VT, and VE in the time course of hypoxic gas exposure.

Metabolic responses to hypoxia during light and dark periods. Figure 2, A–F, shows VO2, VCO2, and R values under normoxic and hypoxic conditions during light and dark periods. In WT mice, VO2 decreased under the hypoxic condition. Circadian variation was seen in both VO2 and VCO2 values irrespective of normoxic or hypoxic conditions. The R value was lowered in the dark period under normoxic conditions. In H1RKO mice, VO2 was decreased under hypoxic conditions. However, the VCO2 level under hypoxic conditions was similar to that under normoxic ones. Circadian changes in VCO2 were not observed in either condition. The R value was increased in the light period under either condition, and circadian changes were clearly exhibited.


Figure 2
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  		Fig. 2. O2 consumption (VO2; A and D), CO2 excretion (VCO2; B and E), and respiratory exchange ratio (C and F) are shown in WT and H1RKO mice. The metabolic variables during the light (open columns) and dark periods (filled columns) are indicated during normoxic and hypoxic gas exposures. Significant main effects of condition and photoperiod were observed for VO2, VCO2, and respiratory exchange ratio. Main effects of genotype were observed for VO2 and respiratory exchange ratio. A significant interaction of the genotype x photoperiod was observed for VCO2 (P < 0.05, 3-way ANOVA). Significant effects of the photoperiod (*P < 0.05, Student's t-test) and hypoxia (#P < 0.05, Student's t-test) were also observed.

 
These findings were confirmed by statistical analyses. The three-way ANOVA yielded significant main effects of condition and photoperiod for VO2, VCO2, and R and significant interactions of the photoperiod x condition for VO2 and VCO2 (P < 0.05). A main effect of genotype for VO2 and R (P < 0.05) and a significant interaction of the genotype x photoperiod for VCO2 (P < 0.05) were also observed. A post hoc test showed that the VCO2 in H1RKO mice was not changed by either condition or photoperiod.

Relationship between VE and VO2. Figure 3 shows the relationship between VE and VO2 during HVD at 10, 15, and 20 min after the commencement of hypoxic gas inhalation. In WT mice, VE decreased as VO2 decreased in the light period, whereas VE was maintained along with VO2 in the dark period. The ratios VE/VO2 at the corresponding times were averaged; these were 0.71 ± 0.02 in the light period and 0.85 ± 0.02 in the dark period. The ratios in the dark period were significantly higher than those in the light period (P < 0.05, Student's t-test). In H1RKO mice, VE decreased as VO2 decreased in both photoperiods. However, the averaged ratios VE/VO2 at the corresponding times were 1.02 ± 0.05 in the light period and 1.04 ± 0.02 in the dark period and were not statistically different. The slopes of the averaged ratio lines were steeper for H1RKO mice than for WT mice in both photoperiods (P < 0.05, Student's t-test), indicating an increase in VE relative to metabolic demand.


Figure 3
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  		Fig. 3. Relationship between VE and VO2 at 10, 15, and 20 min after the commencement of hypoxic gas exposure. Filled symbols indicate values during the dark period, and open symbols indicate values during the light period. Data from WT (circles) and H1RKO mice (triangles) are shown. BW, body weight. Lines indicate the averaged ratios of VE/VO2 in each photoperiod and each genotype. In WT mice, the ratio in the dark period was higher than that in the light period (P < 0.05, Student's t-test); this was not the case in H1RKO mice. The ratios were higher in H1RKO mice than in WT mice in both photoperiods (P < 0.05, Student's t-test).

 
Arterial blood gases. Table 1 shows arterial blood gas values during the light period under normoxic and hypoxic conditions. In the hypoxic condition, PaO2 was decreased in mice of both genotypes (P < 0.05, Student's t-test). PaO2 in the normoxic condition was higher in H1RKO mice than in WT mice. H1RKO mice showed low levels of [HCO3–] with decreases in BE under both normoxic and hypoxic conditions (P < 0.05, Student's t-test).


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  		Table 1. Analysis of arterial blood gases

 
Glucose, lipid, and related substances in blood. As shown in Table 2, analyses of blood showed that the levels of TC, HDL, LDL, and TG in the light periods were lower in H1RKO mice than in WT mice (P < 0.05, Student's t-test). Genotype differences in TC and LDL were observed during the light period. In H1RKO mice, the ketone body level was higher than that in WT mice during the light period (P < 0.05, Student's t-test). The levels of glucose, lactate, and glycated albumin were not significantly different between genotypes and between light and dark periods.


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  		Table 2. Levels of glucose, lipid and related substances in blood

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that during the time course of hypoxia, VE differed between light and dark periods and between genotypes. In WT mice, VE was higher during the dark period than during the light period. However, in H1RKO mice, this normal circadian variation in VE was minimal. In particular, VE was increased relative to metabolic demand during the light period. We clarified the genotype-specific and circadian variations in metabolic rate, blood gases, and blood levels of glucose and lipids that could be attributable to the difference in VE over the time course of hypoxia.

Hypoxic ventilatory response. We observed circadian variation in VE during hypoxia in WT mice. A higher VE during hypoxia during the dark period is consistent with a higher resting VE, metabolism, and body temperature in the same photoperiod in mice and rats (12, 35). Breathing and its control mechanisms accompany daily oscillations in numerous physiological variables (35). Among these variables, metabolism is a major determinant of the change in VE during hypoxia (13), suggesting that a higher metabolic rate may induce an increase in VE during hypoxia in a dark period. Another factor attributable to the circadian variation in VE is a change in carotid chemoreceptor sensitivity. The initial increase in biphasic hypoxic response reflects the activation of carotid chemoreceptors (2). In our study, initial increases in VE did not differ between photoperiods. However, it has been reported that melatonin, having circadian variation in secretion, enhances the hypoxic response of rat carotid body chemoreceptors in vitro (3). Thus studies in respect of carotid chemoreceptor function are needed to better ascertain circadian differences.

In contrast to WT mice, H1RKO mice showed a decrease in the circadian variation in VE over the time course of hypoxia (Fig. 1). Evidence suggests that histamine modulates endogenous circadian rhythms (reviewed in Ref. 42). Histaminergic neurons in the tuberomammillary nucleus are connected to the suprachiasmatic nucleus, which is the pacemaker for circadian rhythms in mammals (27, 37). In experiments performed in free-running conditions in constant darkness, animals followed their internal rhythm without entrainment by light. Under this condition, histamine is involved in phase shifts of circadian activity (1). However, the H1 receptor does not contribute to the circadian rhythm entrained by light in ventilation and metabolism but alters the level of ventilation during the light period (12). In the present study, decreased circadian variation in the VE response to hypoxia depended on a considerable increase in the VE response during the light period. In H1RKO mice, the Dejours test has shown that peripheral chemoreceptor sensitivity is not different from that in WT mice (13), indicating that the genotype difference may be based on central respiratory control rather than peripheral chemoreceptor sensitivity. We then focused on metabolic rate and acid-base status as possible factors producing the genotype difference in VE response to hypoxia, especially during the light period.

Metabolic response to hypoxia. In WT mice, the metabolic rate reached higher values in the dark period than in the light period, consistent with the levels of VE during hypoxia. Additional observations revealed that the circadian variation in metabolism was larger under normoxic conditions than hypoxic ones (significant interaction of photoperiod x condition for both VO2 and VCO2). This result suggested that reduced O2 availability inhibits circadian changes in metabolism, which is in agreement with observations of VO2 in rats (35). The reduced circadian oscillation in VO2 in response to hypoxia is mediated by central mechanisms involving the hypothalamus (7).

There were clear genotype differences in the metabolic rate. H1RKO mice showed diminished circadian changes and hypoxic decline in VCO2, and this was reflected in the R values. Under the normoxic condition, the R value in H1RKO mice was higher than that in WT mice throughout a day, suggesting that the main energy substrates may be altered to make the glycolytic metabolism dominant (12). Histamine H1 receptors in the hypothalamus accelerate lipolysis in brown adipose tissue through the activation of the sympathetic nervous system; therefore, lipolysis is decreased in H1RKO mice (25, 41). These metabolic changes in H1RKO mice would influence R values even under hypoxia, during which R values were significantly increased compared with those of WT mice in the light period.

Relationship between ventilation and metabolic rate during light and dark periods. The VE/VO2 ratio is higher in the active phase in rats during normoxia (35). In mice, a greater VE/VO2 during the dark period is also shown by 24-h monitoring (12). Under hypoxic conditions, VE was increased relative to VO2 during the dark period in WT mice. In addition, VE was maintained even at the end of the 20-min hypoxia, which was associated with the retention of VO2. This profile of ventilation and metabolism in the dark period might be related to a condition in the raised arousal level in the active phase.

In contrast to WT mice, H1RKO mice did not show increases in VE/VO2 during the dark period; however, their VE/VO2 ratios in both light and dark periods were higher than those in WT mice. This inhibition of the increase in the ratio is based on the small value for VE during the dark period relative to the value during the light period, during which emotional and arousal factors might be involved. Intracerebroventricular injection of histamine or H1 receptor agonists induces behavioral arousal (24). Mice lacking histidine decarboxylase (HDCKO), a histamine synthesizing enzyme, show reduced activity levels in wheel running during the dark period; this is associated with a reduction in wakefulness (1). Thus central histamine participates in an arousal level via H1 receptors, whose absence may influence VE/VO2 during the dark period.

On the other hand, VE in H1RKO mice is remarkably increased relative to metabolic demand during the light period, and this should diminish the circadian variation in VE/VO2 (Fig. 3). Arterial blood gases in the light period showed lowered [HCO3–] and BE in H1RKO mice, suggesting that metabolic acidosis occurs in this mouse model (Table 1). Respiratory compensation for metabolic acidosis could possibly occur to increase VE relative to VO2 demand. Arterial blood gases were not evident in the dark period because of technical limitations; therefore, it is uncertain whether the acid-base status is associated with the VE/VO2 ratio in the dark period. Metabolic acidosis is caused by accumulation of fixed acids such as lactate and ketone body in serum. We therefore measured these substances in both genotypes of mice.

Glucose and lipid-related substances in blood. Blood examinations showed high levels of ketone bodies in H1RKO mice, coinciding with the metabolic acidosis resulting from reduced [HCO3–] and BE. Ketone bodies are produced in the liver, mainly from the oxidation of fatty acids, and are exported to peripheral tissues as an energy source during fasting or stress conditions. Ketone bodies are produced and utilized under conditions of hypoglycemia. Histaminergic neurons in the brain are activated by glucoprivation, contributing to the homeostatic control of energy supply in the brain (34). Lowering the glucose level stimulates hypothalamic histamine release, and central administration of histamine induces hyperglycemic action through the sympathetic outflow via H1 receptors (31, 32). These findings show that a lack of H1 receptors may impair the control of glucose homeostasis. In our ad libitum feeding condition, H1RKO mice maintained their levels of glucose and glycated albumin, which are regarded as indicators of the glycemic control state (9). Therefore, glucose intolerance may not account for the ketone body accumulation. Reduced lipid metabolism in H1RKO mice (25) was confirmed by reduced concentration of cholesterols and TG during the light period. Increased levels of ketone bodies may be associated with degradation and/or utilization rather than their generation.

In ad libitum fed animals, ketone body levels in the blood vary throughout a day, increasing during the transition from the light to the dark period (6). In the present study, blood was sampled at 1300 in the light period and at 2200 in the dark period, possibly corresponding to the low and high levels of ketone bodies. The ketone body levels varied between the photoperiods in WT mice; however, in H1RKO mice, the variation was decreased as a result of increased levels during the light period. These results show that ketone body levels might induce metabolic acidosis, introducing an increase in VE and VE/VO2 during hypoxia. In fact, acetazolamide, a potent carbonic anhydrase inhibitor in the kidneys, results in bicarbonaturia and metabolic acidosis, leading to increased VE at high-altitude conditions (22, 40). Furthermore, metabolic acidosis and increased ventilatory drive are supported by a study of acute HCl infusion in rabbits (29). Although the effects of metabolic acidosis on hypoxic response and the circadian profiles of VE remain unknown, the decreased circadian variation in VE during hypoxia is attributable to the acid-base imbalance in H1RKO mice.

Inspired hypoxic gas containing 3% CO2. Inspired hypoxic gas contained 3% CO2 to prevent hypocapnia induced by hypoxic hyperventilation. However, CO2 augments the initial increases in the hypoxic ventilatory response at the level of the carotid body, thus showing an O2-CO2 interaction (21). In an earlier study, we preconditioned mice with 97% O2 + 3% CO2 to prevent an O2-CO2 interaction and then switched to the hypoxic gas with 3% CO2. In that study, H1RKO mice showed a reduction of HVD compared with WT mice (13). In the present study, inhalation gas in a resting state was air and switched to the hypoxic gas containing 3% CO2. This protocol increased the response and might have masked early changes in the HVD. Thus a profile that was inconsistent with our previous study in HVD was observed in H1RKO mice in this study and was probably based on different preconditioning.

In conclusion, we found circadian variations in the changes in VE during acute hypoxia. H1RKO mice showed a prominent increase in VE relative to VO2 during hypoxia in the light period, which diminished the circadian variation in VE during hypoxia. H1RKO mice showed metabolic acidosis during the light period, in which VE was increased as a respiratory compensation during hypoxia. H1 receptors thus play a role in acid-base homeostasis through the control of metabolism and so affect circadian variations in VE during hypoxia in mice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Homma, 2nd Dept. of Physiology, Showa Univ. School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan (e-mail: ihomma{at}med.showa-u.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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H. L. Haas, O. A. Sergeeva, and O. Selbach
Histamine in the Nervous System
Physiol Rev, July 1, 2008; 88(3): 1183 - 1241.
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