Dopaminergic mechanisms of neural plasticity in respiratory control: transgenic approaches.

Data supporting the hypothesis that dopamine-2 receptors (D(2)-R) contribute to time-dependent changes in the hypoxic ventilatory response (HVR) during acclimatization to hypoxia are briefly reviewed. Previous experiments with transgenic animals (D(2)-R 'knockout' mice) support this hypothesis (J. Appl. Physiol. 89 (2000) 1142). However, those experiments could not determine (1) if D(2)-R in the carotid body, the CNS, or both were involved, or (2) if D(2)-R were necessary during the acclimatization to hypoxia versus some time prior to chronic hypoxia, e.g. during a critical period of development. Additional experiments on C57BL/6J mice support the idea that D(2)-R are critical during the period of exposure to hypoxia for normal ventilatory acclimatization. D(2)-R in carotid body chemoreceptors predominate under control conditions to inhibit normoxic ventilation, but excitatory effects of D(2)-R, presumably in the CNS, predominate after acclimatization to hypoxia. The inhibitory effects of D(2)-R in the carotid body are reset to operate primarily under hypoxic conditions in acclimatized rats, thereby optimizing O(2)-sensitivity.

Open-flow plethysmography with pressure-decay compensation.

Whole-body plethysmography is widely used to measure ventilation in awake, unrestrained animals. However, the explicit solution for volumetric analysis of the plethysmograph signal depends upon a closed system, which limits experimental design. Although often used, open-flow plethysmography is complicated by the time-decay of pressure signals generated in the open chamber (e.g. equivalent volume displacements will yield different pressure pulse magnitudes depending upon the rate of application, dP/dt). This problem may be alleviated by first characterizing the time rate of pressure-decay, dP(k)/dt, as a function of pressure magnitude, P, in the plethysmograph, dP(k(P))/dt. Then for each point P(t) in the original signal, subtract the corresponding dP(k(P))(t)/dt from each dP(t)/dt of the original signal to determine the decay-compensated derivative for that point, dP*(t)/dt, and then numerically integrate dP*(t)/dt to generate a pressure-decay compensated signal. The result is a 'virtual closed plethysmograph' trace that enables confident quantitative determination of ventilatory events and volumes with the full advantage of an open-flow plethysmograph.

Ventilatory responses to acute and chronic hypoxia in mice: effects of dopamine D(2) receptors.

We used genetically engineered D(2) receptor-deficient [D(2)-(-/-)] and wild-type [D(2)-(+/+)] mice to test the hypothesis that dopamine D(2) receptors modulate the ventilatory response to acute hypoxia [hypoxic ventilatory response (HVR)] and hypercapnia [hypercapnic ventilatory response (HCVR)] and time-dependent changes in ventilation during chronic hypoxia. HVR was independent of gender in D(2)-(+/+) mice and significantly greater in D(2)-(-/-) than in D(2)-(+/+) female mice. HCVR was significantly greater in female D(2)-(+/+) mice than in male D(2)-(+/+) and was greater in D(2)-(-/-) male mice than in D(2)-(+/+) male mice. Exposure to hypoxia for 2-8 days was studied in male mice only. D(2)-(+/+) mice showed time-dependent increases in "baseline" ventilation (inspired PO(2) = 214 Torr) and hypoxic stimulated ventilation (inspired PO(2) = 70 Torr) after 8 days of acclimatization to hypoxia, but D(2)-(-/-) mice did not. Hence, dopamine D(2) receptors modulate the acute HVR and HCVR in mice in a gender-specific manner and contribute to time-dependent changes in ventilation and the acute HVR during acclimatization to hypoxia.

Increased capillarity in leg muscle of finches living at altitude.

An increased ratio of muscle capillary to fiber number (capillary/fiber number) at altitude has been found in only a few investigations. The highly aerobic pectoralis muscle of finches living at 4,000-m altitude (Leucosticte arctoa; A) was recently shown to have a larger capillary/fiber number and greater contribution of tortuosity and branching to total capillary length than sea-level finches (Carpodacus mexicanus; SL) of the same subfamily (O. Mathieu-Costello, P. J. Agey, L. Wu, J. M. Szewczak, and R. E. MacMillen. Respir. Physiol. 111: 189-199, 1998). To evaluate the role of muscle aerobic capacity on this trait, we examined the less-aerobic leg muscle (deep portion of anterior thigh) in the same birds. We found that, similar to pectoralis, the leg muscle in A finches had a greater capillary/fiber number (1.42 +/- 0.06) than that in SL finches (0.77 +/- 0.05; P < 0.01), but capillary tortuosity and branching were not different. As also found in pectoralis, the resulting larger capillary/fiber surface in A finches was proportional to a greater mitochondrial volume per micrometer of fiber length compared with that in SL finches. These observations, in conjunction with a trend to a greater (rather than smaller) fiber cross-sectional area in A than in SL finches (A: 484 +/- 42, SL: 390 +/- 26 micrometer2, both values at 2.5-micrometer sarcomere length; P = 0.093), support the notion that chronic hypoxia is also a condition in which capillary-to-fiber structure is organized to match the size of the muscle capillary-to-fiber interface to fiber mitochondrial volume rather than to minimize intercapillary O2 diffusion distances.

Apneic oxygen uptake in the torpid pocket mouse Perognathus parvus.

The apneas of many torpid mammals can persist longer than estimated O2 stores allow. This suggests that some O2 is acquired during these apneas by either cutaneous uptake or by a nonventilatory flux down an open airway (tracheal flux). Previous experiments confirmed apneic O2 uptake in the bat Eptesicus fuscus with the conclusion that the uptake most likely occurred by tracheal flux. However, the bat's large cutaneous wing area remained a potential source of cutaneous O2 uptake, leaving uncertainty regarding the mechanism of O2 uptake, particularly in regard to some evidence suggesting that small mammals might be obligated to maintain a closed glottis during apnea. This study sought experimental confirmation of passive O2 uptake in the pocket mouse Perognathus parvus, torpid at a body temperature of 10 degrees C, body mass 16.0 +/- 0.6 g (N = 9). Ventilation bouts lasted 1.49 +/- 0.06 min, whereas apneas lasted 4.51 +/- 0.14 min, despite estimated O2 stores able to support apneas of only 1.0 min. The maximum predicted cutaneous O2 uptake was 0.67 mumol O2/h, whereas the theoretically calculated tracheal flux was 20.2 mumol O2/h. This theoretical rate of tracheal flux compared favorably to the measured plateau apneic O2 uptake rate of 16.7 mumol O2/h. However, the diffusional component of tracheal flux was 3.6 times greater than predicted, indicating an important contribution from cardiogenic mixing. Overall, apneic O2 uptake provided 10.2% of the mouse's total O2 uptake. We conclude that passive tracheal flux is the most likely mechanism by which this animal acquires O2 during apnea.

Increased fiber capillarization in flight muscle of finch at altitude.

We examined fiber capillarization and ultrastructure in the highly aerobic flight muscle of six gray crowned rosy finches (Leucosticte arctoa; mass 22.9 +/- 0.5 (SE) g) living at altitude (A; White Mountains of Eastern California; 4000 m) compared to eight sea-level (SL) house finches (Carpodacus mexicanus, mass, 19.8 +/- 0.6 g) of the same subfamily, Carduelinae. Capillary length per fiber volume (A, 10,400 +/- 409 mm(-2); SL, 7513 +/- 423; P < 0.001) and capillary-to-fiber ratio (A, 2.32 +/- 0.07; SL, 1.85 +/- 0.06; P < 0.001) were significantly greater in A, with no difference in fiber cross-sectional area compared to SL. Capillary geometry was significantly different in A, yielding a greater contribution of tortuosity and branching to capillary length than in SL. Capillary-to-fiber surface ratio and fiber mitochondrial volume were both greater in A, but their ratio was similar to SL, indicating a proportional increase in the size of the capillary to fiber interface and fiber mitochondrial volume in A to sustain high levels of aerobic capacity while living at altitude.

Capillary-fiber geometry in pectoralis muscles of one of the smallest bats.

We previously reported striking similarities in the structural capacity for O2 flux in the highly aerobic flight muscles of a hummingbird and bat despite their significant differences in capillary-fiber geometry and number, and fiber size. However, the bats of that study (Eptesicus fuscus, BW 15-16 g) were about 5 times larger than the hummingbirds (Selasphorus rufus; BW 3-4 g). In this study, we examined the flight muscle in a bat of approximately the same size as the hummingbird to determine whether features found in the big brown bat would be accentuated or if there would be additional similarities with the hummingbird. The pectoralis muscle of pipistrelle bats Pipistrellus hesperus (BW 3-5 g) was perfusion-fixed in situ, processed for electron microscopy and analyzed by morphometry. Fiber size (group mean +/- SE, 314 +/- 22 microns 2 at 2.1 microns sarcomere length) and capillary geometry (high degree of tortuosity and branching) were remarkably similar to those in pectoralis muscle of the big brown bat. Thus distances from capillaries to the center of the fibers were not reduced in pipistrelle flight muscle (as in hummingbird) nor was capillary tortuosity and branching further increased (compared with big brown bat). Capillary-fiber surface ratio at a given mitochondrial volume/microns length of fiber was high and similar to that in big brown bat and hummingbird, consistent with the idea that the size of the capillary-fiber interface plays an important role in providing the great O2 flux potential in these muscles. In addition, capillary-fiber number at a given fiber mitochondrial volume per micron length of fiber was similar to that in other muscles including big brown bat and hummingbird flight muscle, bat hindlimb and rat M. soleus. This supports the notion of a close relationship between capillary number and mitochondrial volume on an individual fiber basis in aerobic muscles.

Apneic oxygen uptake in the torpid bat, Eptesicus fuscus.

Like many mammalian heterotherms, the big brown bat, Eptesicus fuscus, breathes intermittently during torpor. By exploiting this bat's preference to roost in crevices, we could separately measure O2 uptake during ventilatory bouts and apneic periods using a flow-through metabolic chamber with a small dead space volume and short time constant. Oxygen uptake was measured during apneas ranging from 10 to 150 min duration at body temperatures of 20, 10 and 5 degrees C. The fraction of total O2 uptake acquired during apnea was 0.26 +/- 0.03 (9), 0.54 +/- 0.10 (5) and 0.35 +/- 0.04 (3) for body temperatures of 20, 10 and 5 degrees C, respectively. Cardiogenic pulsations during apnea visible on plethysmographic pressure traces and theoretical calculations of airway and cutaneous diffusion potentials support the notion that apneic O2 uptake occurs down an open airway by both diffusion and bulk convection.

Geometry of blood-tissue exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle.

We investigated the relationship between capillary-to-fiber geometry and muscle aerobic capacity by comparing the bat flight muscle (pectoralis muscle), i.e., an ultimate case of extreme O2 demand in a mammalian skeletal muscle, with bat hindlimb and rat soleus muscles. At a given sarcomere length (2.1 microns), fiber cross-sectional area was considerably smaller in bat muscles (pectoralis 318 +/- 10 microns 2, hindlimb 447 +/- 35 microns 2) than in rat soleus muscle (2,027 +/- 125 microns 2). Capillary number per fiber cross-sectional area was much greater in bat pectoralis (6,394 +/- 380/mm2) than in bat hindlimb and rat soleus muscle (2,865 +/- 238 and 1,301 +/- 129/mm2, respectively; all values normalized to 2.1-microns sarcomere length). At the same sarcomere length (2.1 microns), the degree of tortuosity and branching of capillaries were significantly greater in bat pectoralis than in bat hindlimb and rat soleus muscle. In bat flight muscle, capillary length per fiber volume was very high (9,025 +/- 342/mm2). It was 2.2- and 5.4-fold larger than in bat hindlimb and rat soleus, respectively. Mitochondria occupied 35.3 +/- 1.2, 16.5 +/- 1.3, and 6.1 +/- 0.9% of the muscle fiber volume in bat pectoralis, hindlimb, and rat soleus muscles, respectively. There was a strong correlation between capillary length (as well as capillary surface) per fiber volume and mitochondrial volume density in all muscles. Considering capillary supply and mitochondrial volume on an individual fiber basis, we found that 1) the number of capillaries around a fiber was linearly related to mitochondrial volume per micron length of fiber in the muscles but that 2) capillary surface per fiber surface, at given mitochondrial volume per micron length of fiber, was about twice as large in bat pectoralis as in rat soleus muscle, whereas in bat hindlimb it was intermediate between that in bat pectoralis and that in rat soleus muscle. This was due to the differences in fiber size (rat soleus greater than bat muscles) and capillary-to-fiber ratio (bat pectoralis greater than hindlimb) between the muscles. It is notable that in the bat, the substantially greater O2 transfer capacity of the flight muscle compared with hindlimb was achieved by increasing the size of the capillary-to-fiber interface, i.e., capillary-to-fiber surface, via an increase in capillary number rather than by substantially reducing fiber size.

Ventilatory response to hypoxia and hypercapnia in the torpid bat, Eptesicus fuscus.

Ventilatory pattern and ventilatory responses to hypercapnia and hypoxia were investigated in torpid big brown bats at body temperatures of 5, 10, 20, 30 and 37 degrees C. The pattern of breathing at temperatures below 30 degrees C was intermittent, consisting of rhythmic breathing bouts separated by apneic periods with occasional sporadic, non-rhythmic breathing episodes. Overall ventilation (Ve) was matched consistently to overall oxygen consumption (MO2) over the entire range of temperatures with a mean air convection requirement (Ve/MO2) of 1.28 L/mmol. However, calculating the air convection requirement using only oxygen uptake acquired during ventilation yielded an ectotherm-like temperature relationship. Ventilation was stimulated at all temperatures by either increased inspired CO2 or decreased inspired O2. At 20 degrees C, graded hypercapnic stimulation increased the duration of the rhythmic bouts and decreased the duration of apneas until at high CO2 (greater than 3%) breathing was continuous. Hypoxic stimulation below about 7% O2 increased ventilation by selectively increasing the non-rhythmic ventilations and decreasing rhythmic bouts.

Acid-base state and intermittent breathing in the torpid bat, Eptesicus fuscus.

The effects of intermittent breathing on acid-base state and blood gases were characterized in the torpid bat, Eptesicus fuscus, during steady-state torpor between body temperatures (Tb) of 5 and 37 degrees C. Arterial blood samples were taken from indwelling catheters without disturbing the torpid state. Arterial pH (pHa) of samples taken without knowledge of ventilatory state rose by 0.15 units from 37 to 5 degrees C with a delta pHa/delta Tb slope over this range of -0.0055 U/degrees C. However, at and below Tb = 20 degrees C, Eptesicus fuscus breathes intermittently with typical apneic periods of 40-150 min and 4-12 min at 10 and 20 degrees C, respectively. Samples taken at the end of a ventilatory bout and near the end of an apneic period at Tb = 20 degrees C revealed cyclic changes in pH (from 7.49 +/- 0.02 to 7.34 +/- 0.01), PO2 (from 96.6 +/- 3.4 to 30.8 +/- 3.9 Torr), and PCO2 (28.2 +/- 1.4 to 45.9 +/- 1.5 Torr). Between 10 and 37 degrees C, end-ventilatory pHa varied inversely with temperature with a delta pHa/delta T slope of -0.011 U/degrees C. Because intermittent breathing is common to many animals during hibernation, these results demonstrate the importance of coordinating blood sampling with ventilatory state for a reliable interpretation of acid-base regulation under these conditions.

Biological effects of short-term, high-concentration exposure to methyl isocyanate. IV. Influence on the oxygen-binding properties of guinea pig blood.

Whole blood oxygen equilibrium curves (O2 ECs), blood buffer lines, and several hematologic properties were determined for adult guinea pigs exposed to 700 ppm methyl isocyanate (MIC) for 15 min. MIC inhalation effected a significant reduction of blood O2 affinity; the half-saturation pressure (P50) at 38 degrees C increased from the control (untreated) level of 22.8 +/- 0.1 mm Hg to values ranging from 28.5 to 43.7 mm Hg for experimental animals. MIC exposure had no apparent influence on O2 EC shape or CO2 Bohr effect. Erythrocyte volume, [metHb], O2 binding capacity, and combined red cell organic phosphate concentration (DPG + ATP) were not affected by MIC treatment. However, experimental animals experienced a severe metabolic acid-base disturbance; blood lactate concentration ranged from 8.6 to 24.0 mmole/L. Results indicate that lactic acidosis was solely responsible for increased blood P50 of MIC-treated animals. No direct effects of MIC on hemoglobin function were observed. Reduced Hb-O2 affinity, in conjunction with severe hypoxemia, compromised the guinea pigs' capacity for pulmonary O2 loading; at PaO2 of 30 mm Hg, Hb-O2 saturation (S) decreased from 66% S for controls to 42% S for MIC-treated animals.