Selected contribution: variation in acute hypoxic ventilatory response is linked to mouse chromosome 9.

Genetic determinants confer variation among inbred mouse strains with respect to the magnitude and pattern of breathing during acute hypoxic challenge. Specifically, inheritance patterns derived from C3H/HeJ (C3) and C57BL/6J (B6) parental strains suggest that differences in hypoxic ventilatory response (HVR) are controlled by as few as two genes. The present study demonstrates that at least one genetic determinant is located on mouse chromosome 9. This genotype-phenotype association was established by phenotyping 52 B6C3F2 (F2) offspring for HVR characteristics. A genome-wide screen was performed using microsatellite DNA markers (n = 176) polymorphic between C3 and B6 mice. By computing log-likelihood values (LOD scores), linkage analysis compared marker genotypes with minute ventilation (&Vdot;E), tidal volume (VT), and mean inspiratory flow (VT/TI, where TI is inspiratory time) during acute hypoxic challenge (inspired O2 fraction = 0.10, inspired CO2 fraction = 0.03 in N2). A putative quantitative trait locus (QTL) positioned in the vicinity of D9Mit207 was significantly associated with hypoxic VE (LOD = 4.5), VT (LOD = 4.0), and VT/TI (LOD = 5.1). For each of the three HVR characteristics, the putative QTL explained more than 30% of the phenotypic variation among F(2) offspring. In conclusion, this genetic model of differential HVR characteristics demonstrates that a locus approximately 33 centimorgans from the centromere on mouse chromosome 9 confers a substantial proportion of the variance in VE, VT, and VT/TI during acute hypoxic challenge.

Differential lung mechanics are genetically determined in inbred murine strains.

Genetic determinants of lung structure and function have been demonstrated by differential phenotypes among inbred mice strains. For example, previous studies have reported phenotypic variation in baseline ventilatory measurements of standard inbred murine strains as well as segregant and nonsegregant offspring of C3H/HeJ (C3) and C57BL/6J (B6) progenitors. One purpose of the present study is to test the hypothesis that a genetic basis for differential baseline breathing pattern is due to variation in lung mechanical properties. Quasi-static pressure-volume curves were performed on standard and recombinant inbred strains to explore the interactive role of lung mechanics in determination of functional baseline ventilatory outcomes. At airway pressures between 0 and 30 cmH2O, lung volumes are significantly (P < 0.01) greater in C3 mice relative to the B6 and A/J strains. In addition, the B6C3F1/J offspring demonstrate lung mechanical properties significantly (P < 0.01) different from the C3 progenitor but not distinguishable from the B6 progenitor. With the use of recombinant inbred strains derived from C3 and B6 progenitors, cosegregation analysis between inspiratory timing and measurements of lung volume and compliance indicate that strain differences in baseline breathing pattern and pressure-volume relationships are not genetically associated. Although strain differences in lung volume and compliance between C3 and B6 mice are inheritable, this study supports a dissociation between differential inspiratory time at baseline, a trait linked to a putative genomic region on mouse chromosome 3, and differential lung mechanics among C3 and B6 progenitors and their progeny.

Sweating and skin blood flow during exercise: effects of age and maximal oxygen uptake.

Individuals greater than or equal to 60 yr of age are more susceptible to hyperthermia than younger people. However, the mechanisms involved remain unclear. To gain further insight, we examined the heat loss responses of 7 young (24-30 yr) and 13 older (58-74 yr) men during 20 min of cycle exercise [67.5% maximal O2 uptake (VO2max)] in a warm environment (30 degrees C, 55% relative humidity). Forearm blood flow (FBF) and chest sweat rate (SR) were plotted as a function of the weighted average of mean skin and esophageal temperatures [Tes(w)] during exercise. The sensitivity and threshold for each response were defined as the slope and Tes(w) at the onset of the response, respectively. When the young sedentary men were compared with a subgroup (n = 7) of the older physically active men with similar VO2max, the SR and FBF responses of the two groups did not differ significantly. However, when the young men were compared with a subgroup of older sedentary men with a similar maximal O2 pulse, the SR and FBF sensitivities were significantly reduced by 62 and 40%, respectively. These findings suggest that during a short exercise bout either 1) there is no primary effect of aging on heat loss responses but, rather, changes are associated with the age-related decrease in VO2max or 2) the decline in heat loss responses due to aging may be masked by repeated exercise training.

Genetic determinants of acute hypoxic ventilation: patterns of inheritance in mice.

Acutely lowering ambient O(2) tension increases ventilation in many mammalian species, including humans and mice. Inheritance patterns among kinships and between mouse strains suggest that a robust genetic influence determines individual hypoxic ventilatory responses (HVR). Here, we tested specific genetic hypotheses to describe the inheritance patterns of HVR phenotypes among two inbred mouse strains and their segregant and nonsegregant progeny. Using whole body plethysmography, we assessed the magnitude and pattern of ventilation in C3H/HeJ (C3) and C57BL/6J (B6) progenitor strains at baseline and during acute (3-5 min) hypoxic [mild hypercapnic hypoxia, inspired O(2) fraction (FI(O(2))) = 0.10] and normoxic (mild hypercapnic normoxia, FI(O(2)) = 0.21) inspirate challenges in mild hypercapnia (inspired CO(2) fraction = 0.03). First- and second-filial generations and two backcross progeny were also studied to assess response distributions of HVR phenotypes relative to the parental strains. Although the minute ventilation (VE) during hypoxia was comparable between the parental strains, breathing frequency (f) and tidal volume were significantly different; C3 mice demonstrated a slow, deep HVR relative to a rapid, shallow phenotype of B6 mice. The HVR profile in B6C3F(1)/J mice suggested that this offspring class represented a third phenotype, distinguishable from the parental strains. The distribution of HVR among backcross and intercross offspring suggested that the inheritance patterns for f and VE during mild hypercapnic hypoxia are consistent with models that incorporate two genetic determinants. These results further suggest that the quantitative genetic expression of alleles derived from C3 and B6 parental strains interact to significantly attenuate individual HVR in the first- and second-filial generations. In conclusion, the genetic control of HVR in this model was shown to exhibit a relatively simple genetic basis in terms of respiratory timing characteristics.

Hypercapnic ventilatory responses in mice differentially susceptible to acute ozone exposure.

Susceptibility to ozone (O3)-induced pulmonary inflammation is greater in C57BL/6J (B6) than in C3H/HeJ (C3) strain of mice. We tested the hypothesis that altered ventilatory control occurs in B6 mice to a greater extent than in C3 mice after acute O3 exposure. Age-, sex-, and weight-matched C3 and B6 mice were exposed for 3 h to either 2 ppm O3 or filtered air. One and 24 h after O3 or air exposure, whole body plethysmography was used to measure breathing frequency (f), tidal volume (VT), and minute ventilation (VE). To assess changes in ventilatory control, mice were challenged by the elevation of fractional concentration of inspired CO2 levels to 5 and 8% in air for 10 min. After air exposure, there were significantly (P < 0.01) greater changes in VE in B6 than in C3 mice. Hypercapnia-induced changes in VE were significantly (P < 0.01) attenuated in B6 mice 1 h after O3 exposure. VT was significantly (P < 0.01) reduced 1 h after O3 in B6 and C3 mice; however, C3 mice increased f to sustain the hypercapnic VE response similar to air exposure. In contrast, the diminished VT in B6 mice 1 h after O3 occurred coincident with significantly (P < 0.01) reduced f, mean inspiratory flow, and slope of VE-to-%CO2 relationship compared with air exposure. Altered hypercapnic VE in B6 mice was partially reversed 24 h after O3 relative to air-exposed levels. These data suggest that control of ventilation during phenotypic response to CO2 is governed, in part, by genetic factors in inbred strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen replacement in middle-aged women: thermoregulatory responses to exercise in the heat.

Thermoregulatory, cardiovascular, and body fluid responses during exercise in the heat were tested in five middle-aged (48 +/- 2 yr) women before and after 14-23 days of estrogen replacement therapy (ERT). The heat and exercise challenge consisted of a 40-min rest period followed by semirecumbent cycle exercise (approximately 40% maximal O2 uptake) for 60 min. At rest, the ambient temperature was elevated from a thermoneutral (dry bulb temperature 25 degrees C; wet bulb temperature 17.5 degrees C) to a warm humid (dry bulb temperature 36 degrees C; wet bulb temperature 27.5 degrees C) environment. Esophageal (Tes) and rectal (Tre) temperatures were measured to estimate body core temperature while arm blood flow and sweating rate were measured to assess the heat loss response. Mean arterial pressure and heart rate were measured to evaluate the cardiovascular response. Blood samples were analyzed for hematocrit (Hct), hemoglobin ([Hb]), plasma 17 beta-estradiol (E2), progesterone (P4), protein, and electrolyte concentrations. Plasma [E2] was significantly (P < 0.05) elevated by ERT without affecting the plasma [P4] levels. After ERT, Tes and Tre were significantly (P < 0.05) depressed by approximately 0.5 degrees C, and the Tes threshold for the onset of arm blood flow and sweating rate was significantly (P < 0.05) lower during exercise. After ERT, heart rate during exercise was significantly lower (P < 0.05) without notable variation in mean arterial pressure. Isotonic hemodilution occurred with ERT evident by significant (P < 0.05) reductions in Hct and [Hb], whereas plasma tonicity remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypohydration affects forearm vascular conductance independent of heart rate during exercise.

Elevated body core temperature stimulates cutaneous vasodilation, which can be modified by nonthermal factors. To test whether hypohydration affects forearm vascular conductance discretely from relative alterations in heart rate (HR), eight trained cyclists exercised progressively for 20 min each at 60, 120, and 180 W [approximately 22, 37, and 55% of maximal cycling O2 consumption (VO2peak), respectively] in a warm humid environment (dry bulb temperature 30 degrees C; wet bulb temperature 24 degrees C). Esophageal temperature and forearm blood flow were measured every 30 s, and mean arterial pressure and HR were measured at rest and during each exercise intensity (minutes 15, 35, and 55). In the hypovolemic (HP) compared with the euvolemic (EU) state, blood volume was contracted by 24-h fluid restriction an average of 510 ml, and this difference was sustained throughout exercise. The esophageal temperature and HR responses were similar between EU and HP states at 60 and 120 W but were significantly (P < 0.05) higher in HP by the end of 180 W. In contrast, the forearm blood flow response was significantly (P < 0.05) depressed during exercise at 120 and 180 W in HP, whereas mean arterial pressure remained similar between conditions. When body core temperature is elevated in a hypohydrated state, forearm vascular conductance is reduced at exercise intensities of approximately 37% VO2peak, which is independent of relative changes in HR. These findings are consistent with the notion that during exercise an attenuated cutaneous vasodilation is elicited by alterations in regionalized sympathetic outflow, which is unaccompanied by activation of cardiac pacemaker cells.

Differential inspiratory timing is genetically linked to mouse chromosome 3.

Genetic control of differential inspiratory timing (TI) at baseline has been previously demonstrated among inbred mouse strains. The inheritance pattern for TI between C3H/HeJ (C3; 188 +/- 3 ms) and C57BL/6J (B6; 111 +/- 2 ms) progenitors was consistent with a two-gene model. By using the strain distribution pattern for recombinant inbred strains derived from C3 and B6 progenitors, 100% concordance was established between TI phenotypes and DNA markers on mouse chromosome 3. This genotype-phenotype hypothesis was tested by typing 52 B6C3F2 (F2) progeny by using simple sequence repeat DNA markers (n = 21) polymorphic between C3 and B6 strains on mouse chromosome 3. Linkage analysis compared marker genotypes to baseline ventilatory phenotypes by computing log-likelihood values. A putative quantitative trait locus located in proximity to D3Mit119 was significantly associated with baseline TI phenotypes. At the peak (log-likelihood = 3.3), the putative quantitative trait locus determined 25% of the phenotypic variance in TI among F2 progeny. In conclusion, this genetic model of ventilatory characteristics demonstrated an important linkage between differential baseline TI and a candidate genomic region on mouse chromosome 3.

Bloodborne markers in humans during multiday exposure to ozone.

Intermittent exposure of the human lung to ambient levels of ozone (O3) was assayed in systemic fluids by using serum alpha-tocopherol (ST) as a gauge of oxidative stress and the blastogenic activity of peripheral blood monocytes as an index of immune function. Healthy men (n = 10) were evaluated over 3 consecutive days (130 min/day) of chamber exposure to O3 and filtered air (FA); subjects alternated between rest and light treadmill exercise during exposures. For O3, the level was varied at 20-min intervals, i.e., 250, 350, 450, 450, 350, and 250 parts/billion, and concluded with 10 min at 250 parts/billion. ST was quantitated by high-performance liquid chromatography techniques, and T-lymphocyte blastogenesis was measured in cell cultures of peripheral blood monocytes by comparing [3H]thymidine incorporation in mitogen-stimulated (concanavalin A) and nonstimulated cells. After the third day of O3 at 20 h postexposure, ST levels were reduced significantly compared with the FA control subjects (down 14%; -0.96 mumol/l). Mitogen-activated T lymphocytes exhibited a 61% increase in blastogenic activity after 3 days of O3 exposure, significant compared with the proliferative activity of activated T lymphocytes collected after FA or before O3. Acute airway function was impaired by O3, e.g., on day 1, the forced vital capacity and forced expiratory volume in 1 s were decreased 8% (-0.92 liter) and 14% (-0.86 l/s), respectively, from preexposure values, and full recovery was delayed beyond 24 h. Effects of O3 exposure on cellular and biochemical markers increased in magnitude after each exposure and did not parallel the apparent adaptability of bronchial sensitivity to O3.

Leptin attenuates respiratory complications associated with the obese phenotype.

A profile of respiratory complications has been associated with the onset and development of obesity in humans. Similar phenotypes have been routinely demonstrated in genetic animal models of obesity such as the ob mouse (C57BL/6J-Lepob). The objective of the present study was to test the hypothesis that a constellation of respiratory complications are attenuated with leptin (i.e., protein product of the ob gene) replacement. Daily leptin administration during a 6-wk period was conducted to control body weight of mutant ob mice similar to genotypic control groups. During the treatment period, repeated baseline ventilatory measurements were assessed by using whole body plethysmography while quasistatic pressure-volume curves were performed to further explore the role of leptin in improving lung mechanics. Diaphragmatic myosin heavy chain (MHC) isoform phenotype was examined to determine proportional changes in MHC composition. In room air, breathing frequency and minute ventilation were significantly (P < 0.01) different among ob treatment groups, suggesting that leptin opposed the development of a rapid breathing pattern observed in vehicle-treated ob mice. Quasistatic deflation curves indicated that the lung volume of leptin-treated ob mice was significantly (P < 0.05) greater relative to vehicle-treated ob mice at airway pressures between 0 and 30 cmH2O. Diaphragm MHC composition of leptin-treated ob mice was restored significantly (P < 0.05) to resemble the control phenotype. In this genetic mouse model of obesity, the results suggested that respiratory complications associated with the obese phenotype, including rapid breathing pattern at baseline, diminished lung compliance, and abnormal respiratory muscle adaptations, are attenuated with prolonged leptin treatment.

Age and hypohydration independently influence the peripheral vascular response to heat stress.

Seven young (Y, 22-28 yr) and seven middle-aged (MA, 49-60 yr) normotensive men of similar body size, fatness, and maximal oxygen uptake (VO2max) were exposed to a heat challenge in an environmental chamber (48 degrees C, 15% relative humidity). Tests were performed in two hydration states: hydrated (H, 25 ml water/kg body wt 1 h before the test, 2.5 h before exercise) and hypohydrated (Hypo, after 18-20 h of water deprivation). Each test began with a 90-min rest period during which the transiently increased plasma volume and decreased osmolality after drinking in the H condition returned to base line. This period was followed by 30 min of cycle exercise at a mean intensity of 43% VO2max and a 60-min resting recovery period with water ad libitum. Although prior drinking caused no sustained changes in plasma osmolality, Hypo increased plasma osmolality by 7-10 mosmol/kg in both groups. There were no significant age differences in water intake, urine output or osmolality, overall change in body weight, or sweating rate. In the H state, the percent change in plasma volume was less (P less than 0.01) during exercise for the Y group (-5.9 +/- 0.7%) than for the MA group (-9.4 +/- 0.6%). Esophageal temperature (Tes) was higher in the Hypo condition for both groups with no age-related differences. Throughout the 3-h period, mean skin temperature was higher in the Y group and significantly so (P less than 0.05) in the Hypo condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypercapnic duty cycle is an intermediate physiological phenotype linked to mouse chromosome 5.

We hypothesized that upper airway obstruction (UAO) leads to a compensatory increase in the duty cycle [ratio of inspiratory time to respiratory cycle length (Ti/Tt)], which is determined by genetic factors. We examined the compensatory Ti/Tt responses to 1). UAO and hypercapnia among normal individuals and 2). hypercapnia in different inbred strains, C3H/HeJ (C3) and C57BL/6J (B6), and their first- and second-generation (F2) offspring. 3). We then used the compensatory Ti/Tt response in the F2 to determine genetic linkage to the mouse genome. First, normal individuals exhibited a similar increase in the Ti/Tt during periods of hypercapnia (0.11 +/- 0.07) and UAO (0.09 +/- 0.06) compared with unobstructed breathing (P < 0.01). Second, the F2 offspring of C3 and B6 progenitors showed an average Ti/Tt response to 3% CO2 (0.42 +/- 0.005%) that was significantly (P < 0.01) greater than that of the two progenitors. Third, with a peak log of the odds ratio score of 4.4, Ti/Tt responses of F2 offspring are genetically linked to an interval between 58 and 64 centimorgans (cM) on mouse chromosome 5. One gene in the interval, Dagk4 at 57 cM, is polymorphic for C3 and B6 mice. Two other genes, Adrbk2 at 60 cM and Nos1 at 65 cM, have biological plausibility in mechanisms of upper airway patency and chemosensitivity, respectively. In summary, Ti/Tt may serve as an intermediate physiological phenotype for compensatory neuromuscular response mechanisms for maintaining ventilation in the face of UAO and hypoventilation and to help target specific candidate genes that may play a role in the expression of sleep-disordered breathing.

Genetic control of differential baseline breathing pattern.

The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.

Changes in plasma volume during bed rest: effects of menstrual cycle and estrogen administration.

Bed rest (BR) is associated with a decrease in plasma volume (PV), which may contribute to the impaired orthostatic and exercise tolerances seen immediately after BR. The purpose of this study was to determine whether increases in blood estrogen concentration, either during normal menstrual cycles or during exogenous estrogen administration, would attenuate this loss of PV. Nineteen healthy women (21-39 yr of age) completed the study. Twelve women underwent duplicate 11-day BR without estrogen supplementation. PV decreased significantly (P less than or equal to 0.01) during both BR's, from 2,531 +/- 113 to 2,027 +/- 102 ml during BR1 and from 2,445 +/- 115 to 2,244 +/- 96 ml during BR2. The women who began BR in the periovulatory stage of the menstrual cycle (n = 3), a time of elevated endogenous estrogens, had a transient delay in loss of PV during the first 5 days of BR. Women who began BR during other stages of the menstrual cycle (n = 17) showed the established trend to decrease PV primarily during the first few days of BR. Seven additional women underwent a single 12-day BR while taking estrogen supplementation (1.25 mg/day premarin). PV decreased during the first 4-5 days of BR, then returned toward the pre-BR level during the remainder of the BR (pre-BR PV, 2,525 +/- 149 ml; post-BR PV, 2,519 +/- 162 ml). Thus menstrual fluctuations in endogenous estrogens appear to have only small transient effects on the loss of PV during BR, whereas exogenous estrogen supplementation significantly attenuates PV loss.

Fluid restriction prior to cycle exercise: effects on plasma volume and plasma proteins.

The purpose of this study was to test the hypothesis that changes in exercise intensity dominate the PV response to cycle exercise in the heat independent of the initial plasma volume (PV) and total circulating protein (TCP) content. The two experimental treatments (counterbalanced design) were performed by nine trained male cyclists (age = 23 +/- 1 yr, VO2peak = 63 +/- 4 ml.kg-1.min-1) in both a euhydrated (EU) and hypohydrated (HP, 24-h fluid restriction) state. Blood volume was measured (carbon monoxide dilution) 30 min prior to each test and subsequent changes in PV were calculated from serial venous blood samples using hematocrit and hemoglobin concentration. Following 20 min of seated rest in a warm environment (Tdb = 30 degrees C, 50-60% RH), each subject cycled in a semi-reclining posture for 60 min at three successive intensities (60, 120, and 180 W for 20 min each, representing approximately 22, 37, and 53% VO2peak). Fluid restriction reduced (P < 0.05) body weight by 1.4 +/- 0.3 kg (1.8 +/- 0.4%), PV by 353 +/- 73 ml (8 +/- 2%), TCP by 20 +/- 7 g (7 +/- 2%), and elevated serum osmolality by 6 +/- 2 mOsm.kg-1 (2 +/- 1%). After 20 min of passive heat exposure (prior to exercise), TCP content remained lower (P < 0.05) in HP (17 +/- 5 g) compared with EU as PV increased (P < 0.05) in EU (222 +/- 27 ml) but not (P > 0.05) in HP (122 +/- 35 ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Ozone-induced inflammation and altered ventilation in genetically susceptible mice: a comparison of acute and subacute exposures.

We have examined whether the effects of acute (2 ppm/3 h) and subacute (0.3 ppm/72 h) ozone (O3) exposures on airways are mutually predictive. Inbred C57BL/6J (susceptible) and C3H/HeJ (resistant) mice are differentially responsive to inflammation induced by the 2 exposures. Breeding experiments and cosegregation analysis indicated that 2 separate genes control inflammatory responses: Inf (acute), Inf-2 (subacute). The genetic model was also used to examine the effects of both exposures on the magnitude and pattern of breathing. Results imply that mechanisms that control susceptibility to the 2 exposures are not the same, and that one response is not necessarily predictive of the other.

Challenges implicit to gene discovery research in the control of ventilation during hypoxia.

Appointing physiological function to specific genetic determinants requires a systems physiologist to consider ways of assessing precise phenotypic mechanisms. The integration of ventilation, metabolism and thermoregulation, for example, is very complex and may differ among small and large mammalian species. This challenge is particularly applicable to the study of short- and long-term adaptation of these systems to hypoxic exposure associated with high altitude. Our laboratory has initiated a research effort to dissect the complexity of hypoxic adaptation using traditional quantitative genetic analysis and contemporary DNA genotyping techniques. Although the current evidence in murine models demonstrates that specific genes influence control of hypoxic ventilatory responses (HVR), the relevance of these determinants to human adaptation to altitude remains open to exploration. Our review discusses the progress and uncertainties associated with assigning a genetic basis to variation in acute and chronic HVR.

A genomic model for differential hypoxic ventilatory responses.

Inbred mice are routinely used as genetic models in lung biology. Among many phenotypic differences in lung function and structure, C3H/HeJ (C3) and C57BL/6J (B6) inbred mice also demonstrate a significantly different ventilatory pattern during acute hypoxic challenge. The present study rejects the hypothesis that a genomic basis for differential hypoxic ventilatory responses (HVR) is linked to loci which determine differential breathing pattern at baseline, while proposing an alternative genetic model for HVR variation. Twelve BXH recombinant inbred (RI) strains derived from C3 and B6 progenitors were examined to enumerate the genes regulating differential HVR. In each of 134 mice, HVR was assessed using whole-body plethysmography to measure tidal volume (VT) and breathing frequency (f). With respect to f during hypoxia, three distinct and reproducible phenotypes are evident in the BXH RI strain distribution pattern (SDP). The SDP for hypoxic f is consistent with the hypothesis that parental strain differences are regulated by two genes. Cosegregation analysis suggest that the genetic control of f during hypoxia differs from the genes which control differential baseline f. Although the genetic control of VT appears more complex, differences in the minute ventilation (VE) during hypoxia is determined by VT. Therefore, this study suggests that the phenotypic variation in HVR between C3 and B6 parental strains, especially related to f during hypoxia, is regulated by as few as two major genetic determinants.

Mechanisms of response to ozone exposure: the role of mast cells in mice.

Acute and subacute exposure to ozone (O3) induces lung inflammation and hyperpermeability and causes epithelial injury of both upper (nasal) and lower airways. Mast cells are important regulatory cells in mice for each of these effects. Subacute and chronic O3 exposures cause epithelial injury and inflammation in terminal bronchioles and proximal alveoli. Little is known, however, about the mechanisms of injury. Because inflammatory processes may be linked to the pathogenesis of many airway diseases, it is critical to understand the underlying mechanisms that initiate and propagate these processes. We tested the hypothesis that mast cells mediate airway injury induced by chronic O3 exposure by comparing regional airway inflammation and epithelial injury as well as ventilatory responses in genetically mast cell-deficient mice (WBB6F1-KitW/KitW-v [KitW/KitW-v]) with those in (1) normal, mast cell-sufficient, congenic littermates (WBB6F1(-)+/+ [+/+]) and those in (2) KitW/KitW-v mice that were repleted with mast cells by bone marrow transplantation (BMT) from +/+ donors (KitW/KitW-v-BMT). Thus, three (different) groups of mice were used. The following experimental protocol was utilized to test this hypothesis. Animals from each treatment group (n = 4-6/group) were exposed to 0.26 parts per million (ppm) O3 8 hours/day and 5 days/week for durations of 1, 3, 14, 30, and 90 days. Between 8-hour exposures, mice were exposed continuously to 0.06 ppm O3. Age-matched mice were simultaneously exposed to filtered air (0.0 ppm O3) to serve as O3 controls. To evaluate reversibility of exposure-induced lesions, a set of mice from each genotypic group was exposed to air or O3 for 90 days and then placed in HEPA-filtered air for 35 days. After each period of exposure and after 35-day recovery, the nasal cavity and lungs of O3- and air-exposed mice from each group were evaluated for regional inflammation and permeability, epithelial proliferation, and ventilation pattern. Estimates of airway inflammation and hyperpermeability were obtained by analysis of cell differentials and total protein concentrations, respectively, in fluids obtained through use of bronchoalveolar lavage (BAL). Ozone exposure caused significantly greater increases in lung macrophages, epithelial cells, and polymorphonuclear leukocytes (PMNs) in mast cell-sufficient +/+ and KitW/KitW-v-BMT mice than in mast cell-deficient KitW/KitW-v mice. Comparable ozone exposure also elicited increases in lung lymphocytes and in total protein, but there were no significant differences in these two genotypic groups. Cell and total-protein responses in BAL fluid returned to control levels (that is, air exposure only) in all three groups of mice after a 35-day recovery period. The effects of O3 exposure on cell proliferation in the nose and lung were evaluated in the genotypic groups by counting the number of cells that incorporated bromodeoxyuridine (BrdU, a thymidine analog) into DNA. In the centriacinar region of the lung, DNA synthesis was increased significantly in O3-exposed +/+ and KitW/KitW-v-BMT mice, but not in KitW/KitW-v mice, compared with DNA synthesis in air controls. Epithelial proliferation remained significantly elevated or even increased in +/+ and KitW/KitW-v-BMT mice after O3 exposure. Nasal responses to O3 were also evaluated in these three genotypic groups of mice, and there were slight, although statistically significant, O3-exposure effects on the transitional epithelium. However, there were no differences among the groups up to an exposure of 90 days in duration. After a 35-day recovery period, epithelial cell proliferation in +/+ and KitW/KitW-v-BMT mice was greater than that in KitW/KitW-v mice. There were no significant exposure, genotype, or duration effects on baseline ventilation or responses to hypercapnic hypoxia in the three groups of mice exposed to air or O3. (ABSTRACT TRUNCATED)