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Neuroepithelial cells (NECs) of the zebrafish gill filament have been previously identified as bimodal O2 and CO2/H+ sensors that depolarize in response to chemostimuli via inhibition of background K+ channels. To further elucidate the signaling pathway underlying CO2/H+ chemoreception in the NECs we employed microspectrofluorometric techniques to examine the effects of hypercapnia on [Ca2+]i and pHi. NECs increased their [Ca2+]i in response to acidic hypercapnia (5% CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5% CO2, pH 7.8). The acid- induced increase in [Ca2+]i persisted in the absence of extracellular Ca2+, and Ca2+ channel blockers (Cd2+, Ni2+ and nifedipine). NECs exhibited a rapid and reversible drop in pHi in response to acidic hypercapnia and isohydric hypercapnia. Isocapnic acidosis also induced intracellular acidification within NECs, but it was less severe than the drop in pHi elicited by acidic hypercapnia and isohydric hypercapnia. The rate and magnitude of intracellular acidification was reduced by the CA-inhibitor, acetazolamide, without effect on the acid-induced increase in [Ca2+]i. Acetate was used to investigate the relationship between pHi and [Ca2+]i. Acetate induced intracellular acidification without augmentation of [Ca2+]i. The results of this thesis demonstrate that (1) extracellular acidification, but not CO2, is critical to the hypercapnia-induced increase in [Ca2+]i (2) the increase in [Ca2+]i is independent of the drop in pHi (3) the increase in [Ca2+]i is not mediated by the influx of Ca2+ across the plasma membrane.

The purpose of this investigation was to compare the ventilatory threshold (T(vent)) with the lactate threshold (T(lact)) during 60 minutes of steady-state exercise at the calculated thresholds. Eight trained, male cyclists (mean age=23.3 yrs, ht=176.4 cm, wt=70.7 kg, VO₂max=61.02 ml/kg‧minˉ¹) performed a 23 W/min progressive intensity cycling test for determination of T(lact) and T(vent). T(vent) was determined by the non-linear increase in excess CO₂ (ExCO₂) while T(lact) was calculated by the 'individual anaerobic threshold' (IAT) method. Subsequently, subjects performed up to 60 minutes steady-state exercise at the threshold workloads. Results at T(vent) and T(lact) indicate significant differences (p<0.01; T(lact)>T(vent)) between VO₂, ExCO₂, HR, [BLa] and workload as calculated by Hotelling's T²-test. During the steady state exercise at each specified workload, VO₂, [BLa], heart rate and ExCO₂ were measured at 15 minute intervals. All subjects completed the steady-state exercise at T(vent) (VSS) while only 2 subjects completed the steady-state exercise at T(lact) (LSS) (avg time=48.4 min). Comparison of metabolic variables using MANOVA and multiple comparisons revealed significant differences between VSS and LSS for HR and VO₂ at all time intervals, for [BLa] at 30 and 45 minute intervals and for ExCO₂ at the 30 minute interval. Furthermore, examination of [BLa] over time using trend analysis revealed a stabilization during VSS ([formula omitted]=3.05 mmol‧Lˉ¹) whereas [BLa] continuously increased over time during LSS. Findings indicate that T(lact) (IAT method) overestimates the ability to perform prolonged work over 45 min. while T(vent) (ExCO) allows for steady-state exercise greater than 60 minutes. / Education, Faculty of / Curriculum and Pedagogy (EDCP), Department of / Graduate

Two varieties of Pisum sativum, L, Jade and Olympic, were grown for a period of four weeks from seeding, under constant light intensity and diurnally fluctuating temperatures, while being subjected to varying-artificially induced-degrees of nutrient stress. Net carbon dioxide exchange rates were measured in order to determine the influence of excess or deficient nitrogen and phosphorus concentrations within the nutrient medium. Carbon dioxide assimilation rates, under various degrees of nutrient stress were highly variable, both within the same variety and between varieties tested. Microchemical determinations of chlorophylls a and b, and of inorganic phosphate, nitrate and nitrite were carried out in order to determine the relationship of some physiological aspects of mineral deficiency to carbon dioxide assimilation. Investigations as to changes in stomatal index (I) as a result of nutrient stress also showed a large amount of variation. Results obtained indicated that variety Jade was very sensitive to nitrogen deficiency within the rooting medium, as evidenced by a large increase in stomatal index values with nitrogen deficiency. Olympic showed a greater sensitivity to phosphorus deficiency than did Jade. Neither total chlorophyll content, nor chlorophyll a/b ratio appeared to exert any deciding influence upon net carbon dioxide assimilation. A relationship appeared to exist between CO₂ assimilation and quantities of nitrate and/or nitrite present within the photosynthetic tissues of plants tested. This experiment was unable to fully explain the reason(s) for the great degree of variability of net CO₂ exchange rates extant even between plant specimens growing in the same nutrient medium. Some technical improvements applicable to the present experiments as well as some alternative experimental procedures are discussed. / Land and Food Systems, Faculty of / Graduate

The purpose of this study was to examine the changes in the ventilatory (VT) and lactate (LT) thresholds and VO₂max following prolonged aerobic exercise. Six well-trained distance runners (T:age=25.2 yrs, Ht=170.0 cm, Wt=65.0 kg, VO₂max=59.6 ml •kg⁻¹•min⁻¹) and six untrained (UT:age=25.3 yrs, Ht=180.3 cm, Wt=79.2 kg, VO₂max=46.8 ml•kg⁻¹•min⁻¹) males were studied on two occasions seven days apart. The initial evaluation involved a continuous horizontal treadmill test with a starting velocity of 2.22 m•s⁻¹, which was increased by 0.22 m•s⁻¹ each minute until fatigue. Expired gases were continuously sampled and analyzed by a Beckman Metabolic Measurement Cart. Measurements were processed by a data acquisition system (HP 3052A), which determined respiratory gas exchange variables every 15 seconds. Blood lactate measurements were taken via an indwelling catheter during the last 10 sec of each minute of work. VT and LT were determined by visual inspection of the excess CO₂ elimination and lactate curves, respectively. Seven days later the subjects repeated the treadmill test preceded by a 60 minute treadmill run at a heart rate corresponding to their LT. The physiological measurements recorded during the first session were repeated. There were significant (p<0.10) reductions in VO₂max, LT, VT, and total treadmill time on the VO₂max test (TTT) in the T group (59.6 to 56.9 ml •kg⁻¹•min⁻¹, 9.6 to 9.3 mph , 8.9 to 8.2 mph , and 925.0 to 882.5 sec, respectively). VO₂max, LT, VT, and TTT were reduced in the UT group (46.8 to 45.0 ml•kg⁻¹•min⁻¹ 7.7 to 7.6 mph, 8.0 to 7.2 mph, and 730.0 to 652.5 sec, respectively), however, only VT and TTT were reduced significantly (p<0.10). Although the groups were significantly different (p<0.05) in the initial physiological measures due to training status, there was no change in the rate of decline in VO₂max, LT, VT, or TTT when the UT group was compared to T. As LT and VT are affected by prolonged aerobic exercise it is questionable whether these thresholds can be used with confidence to predict endurance performance in events up to 60 min duration for well-trained and recreational athletes. / Education, Faculty of / Curriculum and Pedagogy (EDCP), Department of / Graduate

Oxygen is known to sensitize X-irradiated cells to lethal radiation damage. At low ambient oxygen tensions, however, the molecular mechanisms of the sensitization process and the metabolic requirements of the cell may be forced to compete for the cellular oxygen supply. The effect of cell respiration on the availability of intracellular oxygen during irradiation was consequently investigated by comparing the radiosensitivities of respiring and non-respiring cells. Cultured mammalian cells were irradiated in single cell suspensions and thin film monolayers at respiration inhibiting (4°C) and at normal cell culturing (37°C) temperatures. Due to oxygen equilibration and radiolytic depletion problems, the results of the suspension culture experiments were inconclusive. By subsequently analyzing the diffusive mass transfer of oxygen in the suspension medium, the stirrer flask was determined to be an inappropriate culture vessel in which to irradiate cells at constant low oxygen concentrations. A thin film cell culture system in which the oxygen concentrations to which the cells were exposed during irradiation could be more accurately controlled was then developed. A comparison of the oxygen enhanced radiosensitivities of the respiring and non-respiring cells in thin film monolayers suggested that the metabolic depletion of oxygen at low oxygen tensions has a significant effect on the local and intracellular oxygen distribution. These effects are representative of those that would be produced if respiration induced oxygen gradients existed inside and immediately around respiring cells. The magnitude of the differential radiosensitivities was found to be dependent on cell shape and to have values that agreed very well with theoretical predictions based on the existence of such gradients. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The normal breathing pattern recorded in unanaesthetized, lightly re-strained turtles, Chrysemys picta, consisted of periods of continuous breathing interspersed with periods of breath holding. During each ventilatory period, respiratory frequency and tidal volume were controlled separately and independently of breath length, the total inspiratory interval, the active inspiratory interval and the expiratory interval. Increases in pulmonary minute ventilation during hypercapnia were caused by increases in respiratory frequency due solely to shortening of the intervals of breath holding. The frequency of breathing within each ventilatory period remained constant. There was a large variability in inspiratory and expiratory gas flow rates yet tidal volume was maintained within narrow limits by adjustment of the lengths of the active inspiratory and expiratory intervals. This mechanism was dependent upon lung volume information carried within the vagus nerve. Following vagotomy, changes in minute ventilation due to hypercapnia stemmed primarily from changes in tidal volume while changes in respiratory frequency were markedly reduced. Lung volume information carried within the vagus nerve arose from slowly adapting pulmonary stretch receptors. Single fibre nerve activity from pulmonary receptors was recorded from vagal slips in single-pithed tidally ventilated turtles. The major stimulus of these receptors was the change in lung volume throughout each breathing cycle. The rate and degree of change in transpulmonary pressure were without direct effect on receptor discharge. The functional characteristics of these receptors differed only quantitatively from those recorded in pulmonary stretch receptors of mammals and these differences probably stem from the lower body temperature of the turtle and the location of the receptors in the turtle lung. Most receptors were sensitive to CO₂, several sufficiently sensitive that both tonic and phasic receptor discharge were totally inhibited throughout the ventilatory cycle by 5 to 10% CO₂ in the inspired gas. Pulmonary mechanoreceptors in the frog were also shown to be sensitive to. The acute sensitivity to of a few receptors in turtles and frogs parallels that of the intrapulmonary receptors described-in birds and suggests that a pulmonary receptor with distinct mechano- and chemosensitive properties may represent the functional precursor of the variety of pulmonary receptor types which appear in modern day vertebrates. To examine the role of sensitivity of pulmonary receptors in the overall response of turtles to inhaled CO₂, ventilatory responses of unanaesthetized turtles to changes in the intrapulmonary CO₂ content of a vascularly isolated lung (constant PaCO₂ ) and an intact lung were measured during spontaneous breathing. The isocapnic hyperpnea associated with inhalation of CO₂ by the vascularly isolated lung was small and abolished by vagotomy. It is concluded that both inhibition of pulmonary stretch receptor discharge with increasing levels of FICO₂ and a functional increase in central inspiratory volume threshold contributed significantly to tidal volume increases during hypercapnia. The primary ventilatory response of intact turtles to increasing levels of FICO₂ was an increase in respiratory frequency and this response FICO₂ was greatly reduced when CO₂ was inspired only by the vascularly isolated lung. Thus the ventilatory response of turtles to increasing levels of FICO₂ is primarily dependent upon increased levels of arterial CO₂. The effect of vagotomy in producing apneusis in turtles supports suggestions they lack a pneumotaxic centre. The arrhythmic breathing pattern in turtles with intact vagal nerves, however, bears no similarity to the pattern of breathing in mammals with only the pneumotaxic centre ablated. It is concluded that the vagal input from pulmonary receptors to the respiratory centres in turtles is qualitatively similar to that in mammals yet the differences in central integration of lung volume information in turtles and mammals are not due solely to the absence of a pneumotaxic centre in the turtle. Many of the remaining differences may arise from the lower metabolic demand of turtles but how this affects central integration and respiratory pattern generation is unknown. / Science, Faculty of / Zoology, Department of / Graduate

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