Allan W. Smits

Effects of central and peripheral chemoreceptor stimulation on ventilation in the marine toad, Bufo marinus

The contributions of central and peripheral chemoreceptors to respiratory control in lightly anesthetized Bufo marinus, were assessed by measuring the ventilatory responses to unidirectional ventilation (UDV) of the lungs at several concentrations of CO2 or O2, during intracranial perfusion (ICP) with hypercapnic acidic (5% CO2, pH 7.2) or hypocapnic alkaline (0% CO2, pH 8.3) mock CSF solutions. Peripheral chemoreceptor stimulation alone (hypoxia or hypercapnia during ICP with hypocapnic alkaline CSF) significantly increased breathing frequency and amplitude. ICP with hypercapnic acidic CSF further stimulated ventilation, primarily by significantly increasing the number of breaths/bout of breathing and decreasing the non-ventilatory time at all levels of peripheral ventilatory drive. When peripheral and central chemoreceptor stimulation was low toads were apneic. Stimulation of either central or peripheral chemoreceptors was sufficient to reinitiate breathing. Responses to ICP were greatest when perfusion was directed to the ventral medullary surface (VMS). These results suggest that the initiation of breathing and overall levels of breathing are functions of the combined afferent input from peripheral chemoreceptors and central CO2/pH sensitive chemoreceptors, located near the VMS. Stimulation of central chemoreceptors, however, produced longer duration bouts of rhythmic breathing than did peripheral chemoreceptor stimulation.

The Evolution of the Vertebrate Pulmonary Surfactant System

Lung structure and function vary widely among vertebrates. Despite their diversity, all lungs are internal, fluid-lined structures that change volume and hence face similar biophysical problems. For example, if the surface tension of the fluid lining is high, this may lead to collapse or flooding of the lung In mammals, these problems are largely overcome by the presence of a mixture of surface-active lipids and proteins (pulmonary surfactant), which lowers the surface tension of the fluid lining, particularly at very low lung volumes. This action is due primarily to a disaturated phospholipid (DSP), predominantly dipalmitoylphosphatidylcholine (DPPC), which exists in the ordered, gel state below 41°C Cholesterol (CHOL) and unsaturated phospholipids (USPs) promote respreading upon inflation by converting DPPC to the disordered, liquid-crystalline state. It appeared to us that a DSP-rich surfactant, with its high phase transition temperature, is likely to be of only limited use in the lungs of ectothermic vertebrates that have body temperatures between 20° and 30°C We determined the presence and composition of surfactant in species from a range of vertebrate taxa maintained at 23°C and related variations in phospholipid (PL) head groups, CHOL/PL, DSP/PL, and CHOL/DSP to lung structure and function, phylogeny, and environmental selection pressures such as body temperature. All air breathers examined had a pulmonary surfactant containing USP, DSP, and CHOL. In general, mammals had greater amounts of surfactant lipids than did most nonmammals when expressed per gram of wet lung mass (g WL). However, when expressed per unit of respiratory surface area (cm² RSA), most nonmammalian species tested had six- to 30-fold greater amounts of surfactant lipid than did mammals. Phosphatidylcholine was the predominant PL, and only the minor phospholipids varied between species. We observed surfactant to change in composition from a mixture of very high CHOL/very low DSP in primitive air-breathing actinopterygiian fish, to intermediate CHOL/intermediate DSP in derived lung, flsh and amphibians, to low CHOL/high DSP in reptiles and mammals. We have also observed smaller changes in surfactant composition between species and within individuals, which correlated with dfferences in body temperature, lifestyle, and lung maturity as well as with structure and function of the lung. We determined the pressure required to open a collapsed lung both before and after the removal of surfactant in several species of each vertebrate group and found in virtually all cases that surfactant functioned to lower the lung opening pressure. These findings were consistent with the surfactant functioning as an antiglue in these vertebrate groups. Possibly, acting as an antiglue represents the primitive function of surfactant. On the basis of the two apparently distinct types of surfactant composition, a high CHOL/ low DSP mixture in the primitive air-breathing fish and a mixture of low to intermediate CHOL and intermediate to high DSP levels in the derived sarcopterygiians and the tetrapods, we suggest that the CHOL -enriched surfactant may represent the primitive surfactant, or protosurfactant.

The composition and function of reptilian pulmonary surfactant

In mammals, the surface tension of the fluid lining the inner lung greatly contributes to the work of breathing. Surface tension is modified by the secretion of a mixture of surface active lipids and proteins (termed pulmonary surfactant). A disaturated phospholipid (DSP), predominantly dipalmitoylphosphatidylcholine (DPPC), can eliminate surface tension under high dynamic compression. Cholesterol (CHOL) and unsaturated phospholipids (USP) promote respreading upon inflation by converting DPPC to the disordered liquid-crystalline state. It appeared to us that a surfactant rich in DPPC, which has a high phase transition temperature of 41 degrees C, is likely to be of only limited use in the lungs of reptiles, many of which have preferred body temperatures between 20 and 30 degrees C. We review here the presence and composition of surfactant in species from the three subclasses of the Reptilia and relate these to lung structure and function, phylogeny and environmental selection pressures such as body temperature. We also discuss the function of reptilian surfactant and the factors which control surfactant turnover. Large amounts of pulmonary surfactant have been found in all reptiles so far examined. In general, warmer reptiles have greater amounts of surfactant which is also relatively enriched in DSP. Cold lizards (18 degrees C) have significantly elevated levels of surfactant cholesterol. As in all vertebrates, PC is always the dominant phospholipid (60-80%). Unlike mammals, phosphatidylglycerol (PG) is absent, with the exception of one species. The remaining phospholipid groups are present to varying degrees. The saturated fatty acid, palmitic acid (16:0) is the dominant acyl group, oleic acid (18:1) is the dominant mono-unsaturated fatty acid, and polyunsaturates comprise only about 20% of the total fatty acid profile. For two species of dragon lizards, short term changes in temperature do not affect the fatty acids, but protracted periods of cold significantly decrease the presence of 16:0 in turtle lavage (Lau and Keough, Can.J. Biochem. 59: 208-219, 1981). Surfactant appears to function as an antiglue in most reptiles, serving to lower opening pressure, and decrease the work of breathing. However, surface tension forces generally do not influence reptilian lung compliance, suggesting that the primary functions of mammalian surfactant are not necessarily relevant to reptiles.

Arterial O₂ Homeostasis during Diving in the Turtle Chelodina longicollis

Periodically breathing animals that store O₂ primarily in the lungs must efficiently transfer O₂ to arterial blood during apnea. Previous experiments on diving freshwater turtles indicate that such transferral is continuous during most dives, but there have been occasional observations of much more periodic O₂ transfer between lung gas and blood. This study investigates the dynamics of lung O₂ utilization and blood transfer during voluntary diving in the Australian long-necked turtle, Chelodina longicollis. Pulmonary arterial blood flow was measured by a relatively noninvasive impedance technique. The PO₂ of pulmonary gas and systemic arterial blood was measured continuously with extracorporeal catheter loops, which minimize sampling disturbances. Lung gas PO₂ declined relatively constantly during apnea at a rate of about 3 mmHg/min. Changes in arterial blood PO₂ showed one of two very distinctive patterns during apnea. In the first pattern, evident in about two-thirds of 87 dives monitored in seven turtles, the PO₂ of arterial blood decreased from levels at the start of the dive at a rate of about 1.0-1.5 mmHg/min. In a second pattern observed in the remaining one-third of the dives, arterial PO₂ actually showed periods of transient increase of at least 4 mmHg at some point during the dive, while about 8% showed a transient PO₂ increase of 10 mmHg or more. This second pattern of arterial blood oxygenation was also quite distinctive in that arterial O₂ saturation was maintained constant at around 85%-95%, even when dives lasted for 20 or more min. Qualitative measurements of pulmonary blood flow during voluntary dives indicate that the transient increases in arterial blood PO₂ are closely correlated with large and equally transient increases in pulmonary perfusion. We suggest that C. longicollis can maintain constant arterial O₂ saturation during long periods of diving by periodically increasing pulmonary blood flow to transfer O₂ stored in lung gas into blood perfusing the lungs. Furthermore, we suggest that this may be a general phenomenon among diving reptiles but that its observation requires animals unstressed by sampling techniques.

The Influence of Temperature, Phylogeny, and Lung Structure on the Lipid Composition of Reptilian Pulmonary Surfactant

The lungs of all air-breathing vertebrates contain a form of pulmonary surfactant that lines the alveolar air-water interface where it modifies the interfacial surface tension. These pulmonary surfactants all consist of varying amounts of phospholipids (saturated and unsaturated) and cholesterol. The extent of variation between vertebrate groups and between species within a vertebrate group has been attributed to differences in factors such as phylogeny, body temperature, habitat, and lung structure. The influence of these factors on amphibian surfactant composition and function has been studied, but the reptiles, which comprise a polyphyletic group of vertebrates, have never been critically examined. The surfactant lipid composition from species belonging to the three groups of reptiles, the Archosauria (crocodiles), Lepidosauria (snakes and lizards), and Anapsida (turtles), has been determined. New data is presented in conjunction with already published data to create an evolutionary framework that concentrates particularly on the influence of phylogeny, body temperature, and lung structure on the composition of the surfactant lipids. Large amounts of pulmonary surfactant were found in all species of reptiles. All species lavaged at 23 degrees C (except C. atrox) demonstrated DSP/PL ratios of 23-33%. Animals with multicameral lungs exhibited an elevated CHOL/DSP ratio compared with species with unicameral lungs. In all groups, phosphatidylcholine (PC) was the dominant (60-80%) phospholipid. Phosphatidylserine and phosphatidylinositol (PS/PI) and sphingomyelin (S) represented the other phospholipids, while phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), and phosphatidylethanolamine (PE) were occasionally observed. In two species of lizards (C. nuchalis and P. vitticeps), the saturated fatty acid, palmitic acid (16:0), was the dominant tail group on the phospholipids. Oleic acid (18:1) was the dominant monounsaturated fatty acid, whereas polyunsaturates comprised about a fifth of the total fatty acid profile. Short-term (4 h) changes in temperature did not affect the relative proportions of the fatty acids in either species. Comparison of the current data with previously published literature suggests that phylogeny and habitat do not significantly influence surfactant lipid composition, but body temperature and to a lesser extent lung structure are important determinants of reptilian surfactant lipid composition.

Factors terminating nonventilatory periods in the turtle, Chelydra serpentina

PaO2, PaCO2 and pHa were measured via an extracorporeal loop in conscious snapping turtles (Chelydra serpentina) breathing air or hypoxic (10, 15% O2), hyperoxic (30% O2), or hypercapnic (2% CO2) gases. Turtles breathed into an inverted funnel ventilated with the test gas. Breathing was recorded with a differential pressure transducer. In all turtles, nonventilatory periods were interrupted by breathing episodes containing multiple breaths. In normoxia, PaO2 at the end of nonventilatory periods ranged from 22-128 mm Hg, although PaCO2 showed a less than 5 mm Hg variation about the mean. There was a positive correlation between PaCO2 at the end of the nonventilatory period and the number of breaths in the succeeding period of ventilation. PaCO2 at the end of nonventilatory periods did not change significantly in hyperoxia, although mean PaO2 was significantly increased. In hypoxia, on the other hand, mean PaO2 was significantly reduced and PaCO2 at the end of the nonventilatory period was slightly, but significantly lower. Nonventilatory periods were shorter when turtles breathed 15% O2 (9.3 +/- 1.2 min) or 10% O2 (5.5 +/- 0.3 min) than when they breathed air (17.6 +/- 3.4 min). The results indicate that, in undisturbed turtles, the most important stimulus triggering a breathing episode is the rise in PaCO2 to a critical value during the preceding nonventilatory period. An increase in hypoxic drive shortens the nonventilatory period. However, in normoxia, PaO2 at the end of many nonventilatory periods probably does not fall sufficiently to stimulate O2-sensitive chemoreceptors.

The composition and function of the pulmonary surfactant system during metamorphosis in the tiger salamander Ambystoma tigrinum.

Mammalian lungs secrete a mixture of surface-active lipids (surfactant), which greatly reduces the surface tension of the fluid coating the inner lung surface, thereby reducing the risk of collapse upon deflation and increasing compliance upon inflation. During foetal lung maturation, these lipids become enriched in the primary and active ingredient, a disaturated phopholipid. However, disaturated phospholipids exist in their inactive gellike form at temperatures below 37°C and thus are inappropriate for controlling surface tension in the lungs of many ectotherms. We examined the development of the composition and function of the surfactant system of the tiger salamander (Ambystoma tigrinum) during metamorphosis from the fully aquatic larva (termed stage I) through an intermediate air-breathing larval form (stage IV) to the terrestrial adult (stage VII). Biochemical analysis of lung washings from these three life stages revealed a decrease in the percentage of disaturated phospholipid per total phospholipid (23.03 versus 15.92%) with lung maturity. The relative cholesterol content remained constant. The increased level of phospholipid saturation in the fully aquatic larvae may reflect their generally higher body temperature and the higher external hydrostatic compression forces exerted on the lungs, compared to the terrestrial adults. Opening pressure (pressure required for initial lung opening) prior to lavage decreased from larval to adult salamanders (7.96 versus 4.69 cm H2O), indicating a decrease in resistance to opening with lung development. Opening pressure increased after lavage in older aquatic (stage IV) larvae (5.36 versus 9.80 cm H2O) and in the adults (4.69 versus 7.65 cm H2O), indicating that the surfactant system in salamanders may have an antiglue function which prevents apposing epithelial surfaces from adhering together.

Pulmonary Surfactant Lipids in the Faveolar and Saccular Lung Regions of Snakes

We examined the composition and function of pulmonary surfactant located within the faveolar and saccular regions of the garter snake Thamnophis ordinoides and the rattlesnake Crotalus atrox lung. While the faveolar region is well vascularized and septated and is used in gas exchange, the saccular lung is a thin-walled, smooth bag with very little vascularization that functions as a bellows and in gas storage. Both regions of the lungs of the two snake species contained large amounts of surfactant. The rattlesnake faveolar lung contained 2419.9 ± 260.0 μg of phospholipidper gram of wet lung mass, a value among the largest ever recorded for any species. However, snake faveolar lung surfactant is markedly different from all other vertebrate surfactants reported to date because it contains relatively little cholesterol (3%-8%). Faveolar lung surfactant of the rattlesnake had a greater phospholipid saturation level than that of the garter snake. The phospholipid profile of garter snake faveolar lung surfactant was very similar to that reported for most other nonmammalian vertebrates, with phosphatidylcholine (PC) the predominant phospholipid (64%), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and sphingomyelin (S) either absent or present only in trace amounts (1%-4%), and intermediate (17%) levels ofphosphatidylinositol (PI). The phospholipid profile of rattlesnake faveolar lung surfactant, on the other hand, differed greatlyfrom that of most other vertebrates in that it contained unusually high levels of lysophosphatidylcholine (LPC) and S (12%-18%), whereas PG and PI were virtually absent (0%-1.4%). Removal of surfactant by lavage increased the opening pressure (the initial pressure required to commence the inflation of a collapsed lung) of both regions of the garter snake lung (faveolar region, 1.32-4.51 cm H₂0; saccular region, 1.55-2.19 cm H₂O). However, the rattlesnake faveolar region did not collapse even after lavage, and an opening pressure was never obtained. The opening pressure for the rattlesnake saccular lungs (2.78 cm H₂O) was not increased by lavage (3.0 cm H₂O). The filling pressures were greater for the faveolar lung than for the saccular lung for both species. Filling pressure of the two lung regions was not affected by lavage in either snake.

Surfactant regulates pulmonary fluid balance in reptiles

Reptilian lungs are potentially susceptible to fluid disturbances because they have very high pulmonary fluid filtration rates. In mammals, pulmonary surfactant protects the lung from developing alveolar edema. Reptiles also have an order of magnitude more surfactant per square centimeter of respiratory surface area compared with mammals. We investigated the role of reptilian surfactant 1) in the entry of vascularly derived fluid into the alveolar space of the isolated perfused lizard ( Pogona vitticeps) lung and 2) in the removal of accumulated fluid from the alveolar space of the isolated perfused turtle ( Trachemys scripta) lung by both the pulmonary venous and lymphatic circulations. The flux of fluorescent (fluorescein isothiocyanate) inulin from the vasculature into the alveolar compartment increased 60% after the removal of surfactant, but capillary fluid filtration over a 10-min period was not affected. Surfactant removal decreased alveolar inulin clearance by both the pulmonary venous circulation and the pulmonary lymphatic system ∼1.5- and 3-fold, respectively. In reptiles, fluid flux from capillary to air space must occur indirectly via the interstitium. In the absence of surfactant, this may result in interstitial drying, which affects both pulmonary venous and pulmonary lymphatic clearance of alveolar fluid.

The ventilatory responses of the caecilian Typhlonectes natans to hypoxia and hypercapnia.

Typhlonectes natans empty their lungs in a single extended exhalation and subsequently fill their lungs by using a series of 10–20 inspiratory buccal oscillations. These animals always use this breathing pattern, which effectively separates inspiratory and expiratory airflows, unlike most urodele and anuran amphibians that may use one to many buccal oscillations for lung inflation and typically mix expired and inspired gases. Aquatic hypoxia had no significant effect on the breathing pattern or mechanics in these animals. Aerial hypoxia stimulated ventilatory frequency and increased the number of inspiratory oscillations but had little effect on inspiratory and expiratory tidal volume. Aquatic hypercapnia elicited a large significant increase in air‐breathing frequency and minute ventilation compared to the small stimulation of minute ventilation seen during aerial hypercapnia. Some animals responded to aquatic hypercapnia with a series of three or four closely spaced breaths separated by long nonventilatory periods. Overall, T. natans showed little capacity to modulate expiratory or inspiratory tidal volumes and depended heavily on changing air‐breathing frequency to meet hypoxic and hypercapnic challenges. These responses are different from those of anurans or urodeles studied to date, which modulate both the number of ventilatory oscillations in lung‐inflation cycles and the degree of lung inflation when challenged with peripheral or central chemoreceptor stimulation.