Brain stem preparations from adult turtles were used to determine how bath-applied serotonin (5-HT) alters respiration-related hypoglossal activity in a mature vertebrate. 5-HT (5–20 μM) reversibly decreased integrated burst amplitude by ∼45% (P < 0.05); burst frequency decreased in a dose-dependent manner with 20 μM abolishing bursts in 9 of 13 preparations (P < 0.05). These 5-HT-dependent effects were mimicked by application of a 5-HT1A agonist, but not a 5-HT1B agonist, and were abolished by the broad-spectrum 5-HT antagonist, methiothepin. During 5-HT (20 μM) washout, frequency rebounded to levels above the original baseline for 40 min (P < 0.05) and remained above baseline for 2 h. A 5-HT3 antagonist (tropesitron) blocked the post-5-HT rebound and persistent frequency increase. A 5-HT3 agonist (phenylbiguanide) increased frequency during and after bath application (P < 0.05). When phenylbiguanide was applied to the brain stem of brain stem/spinal cord preparations, there was a persistent frequency increase (P < 0.05), but neither spinal-expiratory nor -inspiratory burst amplitude were altered. The 5-HT3receptor-dependent persistent frequency increase represents a unique model of plasticity in vertebrate rhythm generation.
- control of breathing
- respiratory control
- rhythm generation
serotonin (5-ht) produces complex changes in respiratory motor output. Several 5-HT-receptor subtypes are located pre-and postsynaptically on respiratory neurons in the brain and spinal cord (reviewed in Refs.3, 7, 8, 14,28). One commonly used means of studying the role of 5-HT receptor activation in respiratory motor control is to bath apply serotonergic agonists and antagonists onto reduced in vitro brain stem or brain stem-spinal cord preparations that spontaneously produce respiratory motor output. Most studies using this approach have used in vitro preparations from neonatal rodents (see discussion). Despite the value of information obtained from these experiments, an important caveat is that the distribution of 5-HT-receptor subtypes in respiratory-related regions of the brain stem changes during development (48). Thus it is possible that 5-HT-dependent effects may be different in adult vs. neonatal preparations. In addition, conclusions from studies on neonatal rodent in vitro preparations may be limited when preparations were cooled below physiological temperatures and had regions of hyperoxia, hypoxia, and acidosis (reviewed in Ref. 29).
In this study, we bath applied 5-HT (or serotonergic drugs) onto brain stems isolated from adult turtles. These brain stem preparations are fully mature, remain viable under in vitro conditions for at least 6 h at an appropriate temperature (i.e., room temperature), and produce spontaneous respiratory motor output similar to that produced by intact turtles (11, 19, 20). Although it is possible that turtles may express different 5-HT receptors or serotonergic effects than mammals, we suggest that serotonergic influences must be studied in a diversity of experimental preparations before we will gain a full understanding of serotonergic modulation and its biological significance in respiratory motor control. From a comparative perspective, this is the first report describing the effects of 5-HT on respiratory motor control in an adult poikilothermic vertebrate. Because 5-HT plays a key role in several forms of long-lasting changes in respiratory motor output (reviewed in Refs. 12,28, 40), we determined whether 5-HT induces long-lasting changes in respiratory motor output. A preliminary report of this work was published in abstract form (18).
MATERIALS AND METHODS
Turtle Brain Stem and Brain Stem/Spinal Cord Preparations
Adult turtles (Pseudemys, n = 98, weight = 754 ± 12 g) were obtained from commercial suppliers and kept in a large open tank where they had access to water for swimming and to heat lamps and dry areas for basking. Room temperature was set to 27–28°C with light 14 h/day. Turtles were fed ReptoMin floating food sticks (Tetra, Blacksburg, VA) three to four times per week.
The isolation of the brain stem or brain stem/spinal cord was performed as described previously (19, 20). Briefly, turtles were intubated and anesthetized with 4% halothane or isoflurane in 100% O2 until the limb withdrawal reflex to noxious foot pinch was eliminated. Turtles were then rapidly decapitated and decerebrated. The brain stem or brain stem/spinal cord were removed and pinned down on Sylgard in a recording chamber. The tissue was superfused (4–6 ml/min) with a solution containing HEPES buffer as follows (in mM): 100 NaCl, 23 NaHCO3, 10 glucose, 5 HEPES (sodium salt), 5 HEPES (free acid), 2.5 CaCl2, 2.5 MgCl2, 1.0 K2PO4, and 1.0 KCl. HEPES solution was bubbled with 5% CO2-95% O2 to maintain a pH of ∼7.4, as measured periodically with a calomel glass pH electrode (Digi-Sense, Cole-Parmer, Vernon Hills, IL). In experiments using brain stem/spinal cord preparations, a plastic barrier sealed with petroleum jelly was used to partition the bath into a brain stem compartment (brain stem/spinal cord to spinal segment C2) and a spinal cord compartment (containing spinal segments C3–D1) (20).
To record bursts of respiratory motor activity, glass suction electrodes were attached to hypoglossal nerve rootlets in brain stem preparations (Fig. 1 A) or hypoglossal, pectoralis (expiratory), and serratus (inspiratory) nerves in brain stem/spinal cord preparations as described previously (20). Signals were amplified (×10,000) and band-pass filtered (10–10,000 Hz) by using a differential alternating-current amplifier (model 1700, A-M Systems, Everett, WA) before being rectified and integrated (time constant = 200 ms) by using a moving averager (MA-821/RSP, CWE, Ardmore, PA). Signals were digitized and analyzed by using Axoscope software (Axon Instruments, Foster City, CA).
After the preparations were allowed to equilibrate for 4–6 h, baseline data were obtained by recording 30 min of spontaneous respiratory motor activity before adding drugs to the reservoir. All drugs used in this study were obtained from Sigma/RBI Aldrich (St. Louis, MO) and include: 5-HT, (±)- 8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OH-DPAT; 5-HT1A agonist), 7-trifluoromethyl-4-(4-methyl-1-piperazynil)-pyrrolo[1,2-a]quinoxaline maleate (CGS-12066A; 5-HT1B agonist), methiothepin mesylate (methiothepin; broad-spectrum serotonergic-receptor antagonist that blocks 5-HT1A-F, 5-HT2A, 5-HT5A,5B,5-HT6, and 5-HT7 receptors), (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 5-HT2 agonist); N-phenylimidocarbonimidic diamide (1-phenylbiguanide, 5-HT3 agonist), 3-tropanyl-indole-3-carboxylate hydrochloride (tropesitron, 5-HT3 antagonist).
Data Analysis and Statistics
Respiratory burst variables were measured as described previously (19, 20). Burst amplitude was measured at the highest point of integrated discharge trajectory in arbitrary units, normalized to the average amplitude recorded during the baseline period. Two or more bursts separated by less than the average duration of a single burst were defined as an episode. Because the amplitude of the subsequent bursts may be larger (20), only the first burst of an episode was measured for amplitude data. Burst frequency was calculated as the total number of bursts within a 10-min period. All measurements were averaged into 10-min bins and reported as means ± SE to illustrate the time course of drug effects. For statistical inferences, however, data were combined into 20-min bins and a one-way ANOVA with repeated-measures design (Sigma Stat, Jandel Scientific Software, San Rafael, CA) was used to determine whether data were different from the final 20 min of the baseline period. Combining data into 20-min bins was necessary to reduce the number of groups used in the statistical comparisons. Individual, post hoc comparisons were made by using Dunnett's test. P values < 0.05 were considered significant.
Under control conditions where 5-HT was not applied (n = 10), respiratory amplitude and frequency (5.3 ± 0.2 bursts/10 min) remained constant for at least 3 h (Fig. 1,Ba and Bb). These data are similar to previously published data from isolated turtle brain stems (19).
5-HT Produces Complex Changes in Hypoglossal Respiratory Bursts
5-HT was bath-applied continuously for 60 min at 5, 10, or 20 μM (n = 11, 9, 13 preparations, respectively). During 5-HT applications, hypoglossal amplitude decreased by ∼45% (P < 0.05; Figs. 2 and3) and recovered within 30 min at 5 and 10 μM 5-HT (Figs.2 A and 3, Aa and Ba), but remained depressed for 30 min after 20 μM 5-HT (P < 0.05; Fig. 3 Ca). No 5-HT-induced tonic activity was observed in any experiment. After 2 h of washout, amplitude returned to baseline levels in all experiments.
Five micromolar 5-HT did not alter hypoglossal frequency during application (Figs. 2 A and 3 Ab) but produced a transient doubling of frequency 10 min after washout began (P < 0.05; Fig. 3 Ab). Application of 10 and 20 μM 5-HT decreased mean frequency to nearly zero and abolished bursting for at least 30 min in 3 of 9 and 9 of 13 preparations, respectively (P < 0.05; Figs. 2 B and 3,Bb and Cb). Frequency rapidly returned to levels just above baseline during the washout period after 10 μM 5-HT (Fig.3 Bb). After 20 μM 5-HT, however, frequency overshot baseline levels for 40 min during the washout period (P< 0.05) and remained nonsignificantly elevated by ∼2 bursts/10 min 2 h after washout began (Fig. 3 Cb).
5-HT1A-Receptor Activation Decreases Hypoglossal Burst Amplitude and Frequency
Because 5-HT1-receptor activation decreases hypoglossal burst amplitude in other preparations (38,43), 5-HT1-receptor agonists and antagonists were applied to turtle brain stems. Application of 8-OH-DPAT (5-HT1A agonist) at two different doses for 60 min produced complex changes in amplitude and frequency. Application of 1 μM 8-OH-DPAT (n = 6) did not alter amplitude during drug application, whereas application of 20 μM 8-OH-DPAT (n = 9) decreased amplitude by 50% within 30 min (P < 0.05) and by 20–45% throughout washout (P < 0.05; Fig.4 Aa). Thus long-lasting decreases in amplitude were observed with 5-HT1A-receptor activation.
Frequency was not altered during 1 μM 8-OH-DPAT application but, during washout, increased unevenly with several data points and attained statistical significance (P < 0.05; Fig.4 Ab). During 20 μM 8-OH-DPAT application, respiratory output was abolished for at least 10 min in nine of nine preparations, and frequency decreased to nearly zero before returning to near baseline levels during washout, but these changes were statistically nonsignificant (P = 0.118; Fig. 4 Ab). When 20 μM CGS-12066A (5-HT1B agonist) was applied (n = 7), there were no significant changes in amplitude or frequency compared with baseline (Fig. 4, Ba andBb).
To test whether the 5-HT-dependent decrease in frequency could be blocked by a broad-spectrum 5-HT receptor antagonist, methiothepin (50 μM) was applied for 3.5 h after baseline data were obtained (n = 6). After 30 min of methiothepin (which did not alter amplitude or frequency), 20 μM 5-HT produced no change in amplitude (P > 0.05; Fig. 4 Ca) or frequency during 5-HT application; during washout, however, frequency gradually increased to levels nearly double baseline (P < 0.05; Fig. 4 Cb).
5-HT2-Receptor Activation Does Not Alter Respiratory Activity
To determine which 5-HT-receptor subtypes were responsible for the rebound and long-lasting increase in frequency during 5-HT washout, 20 μM DOI (5-HT2- receptor agonist) was applied (n = 4). After 30 min, amplitude was 1.00 ± 0.04 relative to baseline (P > 0.05) and frequency (6.9 ± 2.9 bursts/10 min) was not changed from baseline (7.6 ± 2.0 bursts/10 min; P > 0.05; data not shown). Thus 5-HT2-receptor activation had no apparent effect on respiratory activity.
5-HT3-Receptor Activation Causes Persistent Increase in Frequency
Phenylbiguanide application (5-HT3 agonist,n = 6) produced a dose-dependent increase in frequency without altering amplitude (Fig.5 A). There was no change in frequency at 1.0 μM phenylbiguanide, a small increase at 10 μM, and a large increase at 20 and 50 μM (3–4 bursts/10 min; Fig.5 B). Because 20 μM phenylbiguanide was the lowest dose that produced a maximal increase in frequency, this dose was used for subsequent experiments. Activation of 5-HT3 receptors with 20 μM phenylbiguanide for 60 min increased frequency by 4–5 bursts/10 min (P < 0.05) and produced a persistent increase of 2–3 bursts/10 min that lasted for at least 2 h during washout (P < 0.05; Fig.6, A and Bb). Amplitude was not altered by phenylbiguanide (P > 0.05; Fig. 6, A and Ba). In separate experiments where 5-HT (20 μM) was applied during a constant background application of tropesitron (5-HT3 antagonist), frequency was abolished for 50 min in eight of nine preparations during 5-HT application (P > 0.05), with no rebound or persistent increase in frequency after 5-HT (Fig. 6 Cb). Amplitude was nonsignificantly decreased by 80% during 5-HT application and returned to ∼70% of baseline (Fig. 6 Ca).
The specificity of tropesitron for 5-HT3 receptors was verified by showing that frequency was unaltered by phenylbiguanide application (20 μM for 1 h) during a constant background tropesitron application (50 μM). In these experiments (n = 2), burst frequency was relatively unaltered from baseline frequency during (increased by 0.8 bursts/10 min) and after phenylbiguanide application (decreased by 0.1 bursts/10 min at 1 and 2 h after phenylbiguanide application) (data not shown). In separate experiments (n = 2) with a constant background tropesitron application (50 μM), 8-OH-DPAT (20 μM for 1 h) nearly abolished frequency and amplitude (data not shown). Thus tropesitron appears to be specific for 5-HT3 receptors in turtles.
Although 5-HT3-receptor activation produced acute and persistent increases in frequency in brain stem preparations without effects on hypoglossal amplitude, excitatory drive to respiratory spinal motoneurons may have been altered. To address this issue, 20 μM phenylbiguanide was selectively applied to the brain stem of brain stem/spinal cord preparations while recording from hypoglossal rootlets and pectoralis (expiratory) and serratus (inspiratory) nerves (n = 5; Fig.7 A). During phenylbiguanide application, hypoglossal, pectoralis, and serratus amplitudes were not significantly changed (Fig. 7, Ac andBa). Frequency increased by ∼2 bursts/10 min during phenylbiguanide application and remained increased by roughly the same amount during a 2 h washout (P < 0.05; Fig.7 Bb).
Although others have investigated the effects of 5-HT on isolated brain stems from neonatal mammals, this is the first study to examine the effects of 5-HT on respiratory activity in an isolated adult vertebrate brain stem preparation. 5-HT application decreased amplitude and frequency in hypoglossal nerve rootlets, an effect likely due to 5-HT1A-receptor activation (although other 5-HT-receptor subtypes may be involved). During 5-HT washout, however, frequency rebounded to levels significantly higher than baseline and remained elevated for at least 2 h. This effect appears to be due to 5-HT3-receptor activation and does not result in alterations in bulbospinal respiratory drive to spinal respiratory motoneurons. To our knowledge, a persistent increase in frequency after 5-HT or 5-HT3 agonist application represents the first example of 5-HT-dependent plasticity in respiratory rhythm generation of a vertebrate under in vitro conditions.
Comparison of Serotonergic and Respiratory Systems in Turtles vs. Mammals
Organization of the turtle (Pseudemys) serotonergic system appears to be similar to that in mammals in several important respects. Turtles have 5-HT-containing neurons along the midline of the brain stem in two nuclei, the nucleus raphe superior and the nucleus raphe inferior (24). Nucleus raphe inferior is probably homologous to the collective raphe pallidus, raphe obscurus, and raphe magnus in mammals. In addition, most areas of the brain stem contain large numbers of 5-HT-immunoreactive fibers (24), suggesting that 5-HT may modulate respiratory rhythm generation and pattern formation at multiple sites. On the other hand, little is known regarding the location and characteristics of respiratory rhythm generating neurons in turtles. Although firing patterns of some turtle brain stem respiratory neurons resemble the firing patterns of mammalian respiratory neurons (47), a comprehensive description of respiratory neurons in turtles is lacking. Thus, despite similarities, one must be cautious when comparing the present results with those expected in adult mammals.
Serotonergic Modulation of Respiratory Motor Output
5-HT effects on respiratory frequency.
5-HT produced complex changes in respiratory frequency, most likely due to the activation of 5-HT-receptor subtypes with opposing influences. During 5-HT application, frequency and amplitude decreased, apparently due to 5-HT1A-receptor activation. After 5-HT application, however, there was a rebound frequency increase that persisted for at least 2 h because of 5-HT3-receptor activation. Blockade of 5-HT1A (and other) receptors (methiothepin) or 5-HT3 receptors (tropesitron) during 5-HT application shifted the balance and allowed expression of the influence from other 5-HT receptor subtypes. Because phenylbiguanide rapidly increased frequency (Figs. 6 Bb and 7 Bb), it is not clear why there was no increase in frequency during 5-HT application with blockade of 5-HT1A and 5-HT1B receptors via methiothepin, which reportedly does not block 5-HT3receptors (Fig. 4 Cb) (16). Perhaps other 5-HT-receptor subtypes not examined in this study (e.g., 5-HT5, 5-HT6, 5-HT7) were activated, thereby opposing the 5-HT3-dependent increase in frequency. Alternatively, methiothepin may unexpectedly block turtle 5-HT3 receptors. Consistent with this hypothesis, phenylbiguanide (20 μM) application to brain stems (n= 3) during a background methiothepin (50 μM) application did not increase burst frequency during or after 1 h of phenylbiguanide application (data not shown). Further experiments will be required to characterize these complex drug interactions.
In several respects, our data from adult turtle brain stems in vitro are directly opposite from in vitro studies on isolated neonatal rat brain stem (and spinal cord) preparations. In those studies, bath-applied 5-HT (and 5-HT-receptor agonists) generally increased respiratory frequency (10, 31, 34, 35), although a biphasic response was reported (39). Likewise, microinjection of 5-HT into the pre-Bötzinger complex of rhythmically active thin slices from neonatal rats increases inspiratory discharge frequency (1). Because respiratory rhythm-generating neurons are postulated to be located in the pre-Bötzinger complex (41, 46), the excitatory influence of 5-HT on frequency in neonatal rat preparations appears to reflect direct actions on respiratory rhythm-generating neurons.
In intact mammals, however, 5-HT exerts a net inhibitory influence on respiratory frequency because 1) systemic or central 5-HT-receptor activation decreases respiration in decerebrate or intact anesthetized cats and rats (2, 27); 2) 5-HT applied to the fourth ventricle in decerebrate cats initially increases respiratory frequency but then decreases frequency to ∼20% below baseline levels for at least 60 min (44); 3) stimulation of raphe neurons abolishes phrenic nerve activity (26); and 4) injection of 8-OH-DPAT into the pre-Bötzinger complex arrests respiratory activity in anesthetized cats (43). Application of 5-HT to the fourth ventricle also decreases respiratory frequency in decerebrate kittens (22) and decerebrate newborn rat pups (23). Thus activation of medullary 5-HT receptors in intact newborn mammals is not necessarily biased toward increased respiratory frequency. Some of these conflicting results, however, are probably due to differences in the method of 5-HT-receptor activation, developmental stage of the animal, species differences, and state dependence of the animal (e.g., awake vs. anesthetized) or overall level of tissue excitability (e.g., producing spontaneous respiratory bursts at a high vs. low frequency).
Role of 5-HT1 receptors.
The role of specific 5-HT- receptor subtypes in respiratory control in turtles is difficult to interpret because at least two receptor subtypes with opposing effects appear to be activated simultaneously during 5-HT application. 5-HT-dependent decrease in frequency and amplitude appears to involve 5-HT1A-receptor activation because 8-OH-DPAT decreased frequency and amplitude in a dose-dependent manner and methiothepin blocked 5-HT-dependent effects. The role of other 5-HT-receptor subtypes cannot be ruled out because methiothepin is a broad-spectrum 5-HT-receptor antagonist (16). 5-HT1A agonists may have activated 5-HT1Aautoreceptors on medullary raphe neurons, decreasing endogenous 5-HT release and thereby augmenting frequency (13, 17, 26). This latter possibility is unlikely because frequency and amplitude were not altered for at least 30 min during the application of methiothepin, suggesting that tonic activity in serotonergic neurons does not modulate frequency under baseline conditions.
Our results are consistent with other studies indicating that 5-HT1A receptor activation in the pre-Bötzinger complex in anesthetized cats depresses phrenic nerve activity in cats (43) and decreases frequency in rhythmically active neonatal rat medullary slices (21). In turtles, there was no evidence for 5-HT1B-receptor activation modulating respiratory rhythm or amplitude (Fig. 4 B).
Role of 5-HT2 receptors.
5-HT2-receptor activation in mammals produces robust increases in respiratory frequency and hypoglossal amplitude (e.g., 1, 39, 44; reviewed in Refs. 3, 14). Thus it was surprising to observe that 5-HT2-receptor activation in turtle brain stems did not alter respiratory motor output (activation of spinal 5-HT2 receptors in turtle brain stem/spinal cord preparations also does not depolarize spinal motoneurons; S.M. Johnson and G. S. Mitchell, unpublished observations). This lack of responsiveness to 5-HT2- receptor activation represents a unique fundamental difference in turtles compared with other vertebrates. Consequently, turtle respiratory neural control system probably does not express 5-HT2-dependent forms of respiratory plasticity that have been described in mammals (reviewed in Refs. 12, 28).
Role of 5-HT3 receptors.
In turtle preparations, activation of brain stem 5-HT3receptors produced a persistent increase in frequency, without significantly altering the amplitude of spinal expiratory and inspiratory motor output. This suggests that brain stem 5-HT3-receptor activation should elicit a long-lasting increase in ventilation in intact turtles. This is the first study to show that 5-HT3-receptor activation in a vertebrate brain stem alters respiratory frequency. In previous studies on in vitro neonatal rat preparations, application of 5-HT3-receptor antagonists did not alter the response to 5-HT (1, 34). These results may differ because of species (turtle vs. rat) or developmental differences (adult vs. neonate).
The mechanisms underlying 5-HT3-dependent persistent frequency increase are not known but are nevertheless intriguing because 5-HT3 receptors are ligand-gated ion channels (16). Thus 5-HT3-receptor activation would be expected to have rapid, brief effects on respiratory motor output, unlike other metabotropic 5-HT-receptor subtypes. One hypothesis is that 5-HT3- receptor activation depolarizes respiratory neurons sufficiently to allow calcium influx via voltage-dependent, membrane-bound calcium channels, thereby increasing intracellular calcium and activating second- messenger systems that induce long-lasting changes in neuronal membrane properties or synaptic transmission. Such changes may, in time, produce a persistent frequency increase. Alternatively, 5-HT3 receptors may be located on neuromodulatory neurons within the brain stem that project to respiratory neurons. Thus 5-HT3-receptor activation may cause depolarization and release of unspecified neuromodulator(s) onto respiratory neurons, subsequently producing long-lasting changes in the turtle respiratory control network.
5-HT effects on hypoglossal burst amplitude.
In general, 5-HT-receptor activation increases hypoglossal motoneuron excitability in a wide range of in vitro and in vivo experimental preparations (1, 4-6, 21-23, 25, 30, 36, 42, 44,48). Increased excitability of hypoglossal motoneurons in mammalian preparations may be due to a combination of 1) direct excitation via postsynaptic 5-HT2 receptors (4, 6, 25, 30), 2) decreased action potential after hyperpolarization via postsynaptic 5-HT1A receptors (48), 3) decreased glycinergic synaptic transmission via presynaptic 5-HT1B receptors (48), or 4) increased transmission between rhythm-generating circuits and motoneurons. In contrast, 5-HT reduced hypoglossal amplitude by 40–50% in turtles, a finding consistent with other studies demonstrating a 5-HT-dependent decrease in hypoglossal nerve activity in neonatal rat brain stems (15, 33,35-37).
Alternatively, 5-HT-dependent decrease in amplitude in turtles may have been due to activation of presynaptic 5-HT1A or 5-HT1B receptors, decreasing glutamatergic inspiratory drive to hypoglossal motoneurons (4, 21, 38, 45). Our data suggest that 5-HT-dependent amplitude decrease was due to 5-HT1A-receptor activation, although other 5-HT-receptor subtypes may be involved. There was no consistent change in amplitude in the present study after 5-HT3- receptor activation or blockade despite evidence for 5-HT3 receptors in the hypoglossal motor nucleus of rats (32, 38).
Functional Significance of 5-HT and Respiratory Motor Control in Turtles
The physiological conditions that cause the release of 5-HT onto respiratory-related neurons in turtles (and reptiles) are not known. One possibility is that 5-HT may be released at the onset of diving behavior to activate 5-HT1A receptors and temporarily minimize ventilatory drive. On return to the surface, 5-HT release would end, and then a 5-HT3-dependent mechanism would augment postdive ventilation to rapidly restore blood gas homeostasis. Such a pattern of increased ventilation due to increased frequency with minor changes in tidal volume is observed in turtles after prolonged anoxia (9).
The authors thank K. B. Bach for the drawing in Fig. 1 and T. L. Baker for critical comments.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-60028 and HL-36780.
Address for reprint requests and other correspondence: S. M. Johnson, Dept. of Comparative Biosciences, School of Veterinary Medicine, Univ. of Wisconsin, 2015 Linden Dr. West, Madison, WI 53706 (E-mail:).
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- Copyright © 2001 the American Physiological Society