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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
1Department of Zoophysiology, Aarhus University, Aarhus, Denmark; 2School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom; 3 Departamento de Zoologia, Centro de Aquicultura, Universidad Estadual Paulista, Rio Claro, SÂo Paulo, Brazil; and 4Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates
Submitted 22 June 2004 ; accepted in final form 13 October 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Val and Ser6 Thr) compared with mammalian BK. Bolus intra-arterial injections of synthetic rattlesnake BK (0.01–10 nmol/kg) into the anesthetized rattlesnake, Crotalus durissus terrificus, produced a pronounced and concentration-dependent increase in systemic vascular conductance (Gsys). This caused a fall in systemic arterial blood pressure (Psys) and an increase in blood flow. Heart rate and stroke volume also increased. This primary response was followed by a significant rise in Psys and pronounced tachycardia (secondary response). Pretreatment with NG-nitro-L-arginine methyl ester reduced the NK-induced systemic vasodilatation, indicating that the effect is mediated through increased NO synthesis. The tachycardia associated with the late primary and secondary response to BK was abolished with propranolol and the systemic vasodilatation produced in the primary phase was also significantly attenuated by pretreatment, indicating that the responses are caused, at least in part, by release of cathecholamines and subsequent stimulation of 
-adrenergic receptors. In contrast, the pulmonary circulation was relatively unresponsive to BK. reptile; vasoactive kinin; catecholamines; nitric oxide; adrenergic receptor
Bradykinin is generated in the blood by the kallikrein-kinin system and involves sequential action of a series of proteolytic enzymes. Activation of mammalian factor XII (Hageman factor) at the site of tissue injury catalyzes plasma prekallikrein and generates BK by cleavage of high-molecular-mass kininogen (2). BK can also be produced from kininogens in vitro by incubation of plasma with a charged surface such as glass beads or by trypsinization (10). BK is rapidly degraded, primarily in the pulmonary circulation.
The blood of reptiles contains some, but not necessarily all, components of the kallikrein-kinin system present in mammals. For example, BK is not generated from the plasma of lizards and snakes through contact with a charged surface, which indicates their blood does not contain a component analogous to mammalian factor XII. BK has, nevertheless, been generated from the plasma of all major groups of reptiles, including several lizards and snakes, but the primary structure differs considerably (reviewed in 6).
In the present study, we describe the purification and structural characterization of BK from a representative of the highly evolved Viperidae family of snakes, the diamondback rattlesnake, C. atrox, from North America. We then investigate the effects of bolus intra-arterial injections of a synthetic replicate of rattlesnake BK on the systemic and pulmonary circulations of anesthetized specimens of the South American rattlesnake, C. durissus terrificus. C. durissus terrificus and C. atrox are relatively closely related within the genus of rattlesnakes (20). Given their close phylogenetic relationship and the observation that the structure of rattlesnake BK is identical to that of other derived snakes (15), the potential problems of characterizing the cardiovascular effects of BK from Crotalus atrox in Crotalus durissus are not likely to affect the validity of our study.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of the kinin. A sample (11 ml) of heparinized plasma from a single specimen of the diamondback rattlesnake (C. atrox) was provided by Dr. Valentine A. Lance (Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA) and was stored at –70°C. Plasma was diluted 10-fold with 0.2% (vol/vol) acetic acid and heated on a boiling water bath for 30 min. After cooling to 25°C, pH was adjusted to 7.8 by addition of 0.5 M Tris·HCl. The denatured plasma was incubated with 1-tosylamide-2-phenylethylchloro-methyl ketone-treated trypsin (Sigma, St. Louis, MO) (6 mg) for 30 min at 37°C and centrifuged (1600 g for 20 min). Peptide material was isolated from the supernatant using Sep-Pak C18 cartridges as previously described (7). Bound material was eluted with 70% (vol/vol) acetonitrile/water and lyophilized.
Radioimmunoassay. BK-like immunoreactivity (BK-LI) was measured using antiserum BT4, as previously described (15). The antiserum was raised against mammalian BK in rabbits and the epitope in BK recognized by the antiserum involves contributions from residues 3, 5, 6, and 9.
Purification of the kinin. The trypsin-treated plasma extract, after partial purification on Sep-Pak cartridges, was redissolved in 1% (vol/vol) trifluoroacetic acid/water (3 ml) and chromatographed on a 90 x 1.6 cm column of Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 24 ml/h, and fractions were collected. Absorbance was measured at 280 nm. Aliquots of the fractions (100 µl) were lyophilized and reconstituted in the same volume of assay buffer [0.05 M sodium phosphate, pH 7.4 containing 0.4% (wt/vol) BSA]. The concentration of BK-LI in the fractions was determined by radioimmunoassay. Fractions containing BK-LI were pooled and injected onto a 25 x 1-cm Vydac 218TP510 C18 reverse-phase HPLC column (Separations Group, Hesperia CA) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 14% over 10 min and to 35% over 50 min using linear gradients. Absorbance was monitored at 214 and 280 nm and fractions (1 min) were collected. The fraction containing BK-LI was rechromatographed on a 25 x 0.46-cm Vydac 214TP54 (C4) column equilibrated with acetonitrile/water/trifluoroacetic acid (10.5/89.4/0.1) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 38% over 40 min using a linear gradient. Rattlesnake BK was purified to near homogeneity by successive chromatographies on 25 x 0.46 cm Vydac 219TP54 (phenyl) and Vydac 218TP54 C18 columns. The concentration of acetonitrile in the eluting solvent was raised from 7 to 35% over 40 min, and the flow rate was 1.5 ml/min.
Structural analysis. The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 491 Procise sequenator (Foster City, CA). Mass spectrometry was performed on a Perkin Elmer Sciex API 150EX single-quadrupole instrument. The accuracy of mass determinations was ±0.02%.
Peptide synthesis. Rattlesnake BK (Val-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was synthesized by solid-phase methodology on a 0.025 mmol scale using an Applied Biosystems model 432 synthesizer. Fluorenylmethoxy-carbonyl-labeled amino acids were coupled as their hydroxybenzotriazole-active esters, following the manufacturer's standard protocols. The peptide was cleaved from the resin using trifluoroacetic acid/water/ethanedithol/thioanisol (900/30/30/40 by vol) and purified by reverse-phase HPLC under the conditions shown in Fig. 1A. The peptide was >95% pure, and the correct sequence was confirmed by automated Edman degradation and electrospray mass spectrometry (observed mass 1,016.4 Da, calculated mass 1,016.6 Da).
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Experimental animals. Studies were undertaken on the South American rattlesnake, C. durissus terrificus of both sexes. Snakes were obtained from the Butantan Institute in São Paulo and transported to Universidad Estadual Paulista, Rio Claro, São Paulo (Brazil), where they were housed in a 0.5 x 0.5 m vivariums, and maintained under natural photoperiod at 28 ± 5°C. This study was conducted in accordance with the "APS Guiding Principles for Research Involving Animals and Human Beings." The snakes had free access to water, but food was withheld 1 wk before experimentation. All animals appeared healthy, with body masses ranging from 0.27 to 0.95 kg (0.61 ± 0.06 kg).
Surgery and instrumentation. Snakes were anesthetized by an injection of 30 mg/kg pentobarbital sodium into the tail muscle (Mebumal, Sygehusapotkerne, Denmark). All reflexes disappeared within 20 min, and the animals were then placed in a prone position, so that they could be traecheotomized for artificial ventilation at 4 breaths/min and a tidal volume of 25 ml/kg using a Harvard Apparatus mechanical ventilator. A 5-cm ventral incision was made cranial to the heart, and a PE-50 catheter containing heparinized saline was advanced into the right aortic arch through the vertebral artery. An additional incision was made immediately above the heart. A small branch of the pulmonary artery supplying the dorsal part of the lung was occlusively cannulated with a PE-50 catheter containing heparinized saline. Both catheters were connected to Baxter Edward (model PX600, Irvine, CA) disposable pressure transducers, and the signals were amplified using an in-house built preamplifier. The pressure transducers were positioned at the level of the heart of the snake and were calibrated daily against a static water column.
For measurements of blood flows, 2S or 2R transit-time ultrasonic blood flow probes (Transonic System, Ithaca, NY) were placed around the left aortic arch (LAo) and the pulmonary artery. Acoustical gel was infused around the blood flow probes to enhance the signal. Both flow probes were connected to a Transonic dual-channel blood flowmeter (T206). Signals from the pressure transducers and the blood flowmeter were recorded with a Biopac MP100 data-acquisition system (Biopac Systems, Goleta, CA) at 50 Hz.
Calculation of blood flows, stroke volume, and vascular resistances. Because there is only one pulmonary artery in the rattlesnake, measurements of blood flow in the pulmonary artery represent total pulmonary blood flow (Qpul). Total systemic blood flow (Qsys) can be estimated as 3.3 times blood flow in left aortic arch (QLAo) (Galli, Skovgaard, Abe, Taylor, Conlon, and Wang, unpublished observations). Total cardiac output (Qtot) was calculated as Qsys + Qpul. Heart rate (fH) was derived from the instantaneous blood flow trace from the left aortic arch and total stroke volume (Vs,tot; pulmonary + systemic) was calculated as Qtot/fH. When baseline blood flow changes more than baseline blood pressure, which is the case in most in vivo situations, calculated values of conductance provide a better index for comparing vascular tone than resistance (14, 22). Pulmonary and systemic conductance (Gpul and Gsys, respectively) were calculated from mean blood flow and mean blood pressure (Gpul = Qpul/Ppul and Gsys = Qsys/Psys), assuming that central venous blood pressures were negligible.
Experimental protocol. When all hemodynamic parameters had stabilized after surgery, a 0.1 ml/kg injection of 0.9% (wt/vol) saline containing 0.5% (wt/vol) bovine serum was given as a sham injection. Eight snakes then received a series of bolus injections of increasing doses of rattlesnake BK (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 nmol/kg). Hemodynamic variables were allowed to return to baseline levels between each injection.
To investigate the mechanism of action of BK, the effects of treating the snakes with the -adrenergic receptor antagonist propranolol or the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) were studied. In nine snakes, the following drugs were injected into the arterial catheter in the following order: BK (0.3 nmol/kg), epinephrine (2 µg/kg), propranolol (2 mg/kg), epinephrine (2 µg/kg), and BK (0.3 nmol/kg). The injections of epinephrine were performed to assess the effectiveness of adrenergic blockade, and propranolol was allowed to take effect for 20 min before subsequent injections. In a further eight snakes, the effects of BK (1 nmol/kg) before and after treatment with the nitric oxide synthase inhibitor L-NAME (150 mg/kg) were studied.
Data analysis and statistics. All data are presented as means ± SE. Recordings of blood flows and pressures were analyzed using AcqKnowledge data-analysis software (version 3.23; Biopac, Goleta, CA). Effects on hemodynamic variables were assessed by a one-way ANOVA for repeated measurements followed by a post hoc test to identify values that were significantly different from control values. A limit for significance of P < 0.05 was applied.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of rattlesnake BK.
The BK-LI in the trypsin-treated rattlesnake plasma, after partial purification on Sep-Pak cartridges, was eluted as a broad, single peak with maximum immunoreactivity at the elution volume of mammalian BK. The immunoreactive fractions were pooled and injected onto a semipreparative Vydac C-18 HPLC column (Fig. 1A). BK-LI was eluted in fractions shown by the bar (Fig. 1A). These fractions were pooled and chromatographed on an analytical Vydac C4 column (Fig. 1B). The material was heterogeneous, and the BK-LI was associated with the well-defined peak, denoted by arrowheads. Further purification was accomplished on an analytical Vydac phenyl column (Fig. 1C), and the BK-LI was associated with the sharp peak, denoted by arrowheads. A final chromatography of rattlesnake BK on analytical Vydac C-18 column (Fig. 1D) demonstrated that the peptide had been purified to apparent homogeneity, as indicated by the sharp, symmetrical nature of the peak. The final yield of purified peptide was 
2 nmol.
Structural characterization of rattlesnake BK. The primary structure of rattlesnake BK was established without ambiguity by automated Edman degradation as Val-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg. The amino acid composition of the peptide [found: Gly 1.2 (1), Arg 1.1 (1), Thr 1.2 (1), Pro 2.6 (3), Val 1.0 (1), Phe 1.8 (2) residues/mol peptide] is consistent with the proposed structure. Values in parentheses show the number of residues predicted from the sequence analysis data. The structure was also confirmed by mass spectrometry (observed molecular mass 1,016.5 Da; calculated molecular mass 1,016.6 Da).
Hemodynamic Effects of BK in Anesthetized Crotalus
Cardiovascular effects of rattlesnake BK.
Figure 2 shows the recorded hemodynamic changes after a bolus injection of 0.3 nmol/kg synthetic rattlesnake BK. BK produced a rapid (within 30 s) and significant fall in Psys that was accompanied by an immediate (within 15 s) increase in Qsys, while fH increased gradually at a slower rate. This primary phase of the response lasted for 120 s and was followed by a further increase in fH and a secondary increase in Psys as the vasodilatation subsided and Gsys returned to preinjection values (secondary phase). The two phases were also evident in the pulmonary circulation, but the change in pressures and flows were much smaller than observed in the systemic circulation. The biphasic response depicted in Fig. 2 was present at all dosages of BK, but the magnitude and duration of the changes increased with dose. The mean maximal changes in hemodynamic variables during the primary phase are shown in Figs. 3 and 4, while the mean maximal changes during the secondary response are illustrated in Figs. 5 and 6. There were no effects of administrating the vehicle.
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There was also a clear dose-dependency of the responses during the secondary phase (Figs. 5 and 6). A significant rise in Psys occurred at 3 nmol/kg, while Qsys significantly increased at 1 and 3 nmol/kg (Figs. 5, A–C). There were no significant changes in the pulmonary circulation (Figs. 5, D–F). Doses of BK above 0.1 nmol/kg produced a progressive increase in fH, while Vs,tot decreased at the highest dose (3 nmol/kg; Fig. 6, A and B).
To assess whether the systemic vasodilatation during the primary phase and the tachycardia during the secondary phase were -adrenergically mediated, we injected the -adrenergic receptor antagonist propranolol. Injection of propranolol caused significant reductions in both systemic and pulmonary pressures and flows, and heart rate decreased significantly (Table 1). Gsys remained relatively constant after propranolol treatment, while Gpul decreased significantly. Propranolol completely abolished the effect of epinephrine on fH, which indicates that the -adrenergic receptors were fully blocked (Table 1). The effect of epinephrine before and after injection of propranolol is shown in Table 1. In the systemic circulation, epinephrine caused a significant decrease in Gsys, which was of similar magnitude before and after treatment with propranolol (Table 1). In the pulmonary circulation, epinephrine caused a substantial vasodilatation. After treatment with propranolol, however, epinephrine produced a small decrease in Gpul. The rise in Gsys caused by BK (0.3 nmol/kg) was significantly attenuated after treatment with propranolol (Fig. 7). The significant tachycardia observed during the primary and secondary phase of the response was abolished after -adrenergic blockade.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-adrenergic receptors. In addition, pretreatment with L-NAME attenuated the systemic vasodilatation, indicating that increased NO synthesis is also a contributing factor. Our study was performed on anesthetized animals. Anesthesia leads to a higher fH and Qpul compared with fully recovered Crotalus, which appears to be caused by a withdrawal of vagal tone and elevated sympathetic activity (Wang, Taylor, and Abe, unpublished observation), while autonomic responses, such as barostatic regulation, are virtually abolished. The depressed autonomic function is advantageous for studies on local regulation of the cardiovascular system because there are no reflex responses to altered blood pressure.
Structural Characterization of BK
The bradykinin-related peptide ([Val1,Thr6]BK) generated from Crotalus atrox, which belongs to the highly evolved family of viperid snakes, was identical to the BK-related peptide from colubrid snakes (15). However, it contained one amino acid substitution (Ala1 Val) compared with BK from the reticulated python, which is regarded as being relatively primitive (7). The primary structure of BK also varies among other groups of reptiles (reviewed in 6). Treatment of plasma from the alligator (A. mississipiensis) and the turtle (T. scripta) with glass beads in the presence of a kininase inhibitor generated [Thr6]BK (4, 8). In contrast, all attempts to generate BK in the plasma of lizards and snakes through contact with a charged surface have been unsuccessful, suggesting that their blood does not contain a component analogous to mammalian factor XII. However, treatment of heat-denatured plasma from the monitor lizard Varanus grayi with trypsin generated [Thr6]BK, whereas similar treatment of plasma from the Gila monster, Heloderma suspectum generated [Leu2,Thr6]BK (15).
Cardiovascular Responses to BK
Infusion of BK into rattlesnakes caused an initial vasodilatation of the systemic circulation that was manifested as a fall in systemic arterial pressure and a rise in Qsys. This primary response was followed by a subsequent rise in Psys as fH increased and Gsys returned to baseline values. These responses closely resemble the biphasic response of mammals but differ somewhat from other species of reptiles. Turtles and pythons also exhibit a systemic vasodilatation when injected with their native BK, but the changes in blood pressure and heart rate differ. Turtles had a monophasic response, where the vasodilatation is associated with a fall in Qsys, while Psys remained constant (8). There was, however, no tachycardia associated with BK injection in turtles. In pythons, BK also caused a pronounced vasodilatation, but Psys increased as a result of a profound tachycardia and a rise in Qsys (28).
The BK-induced systemic vasodilatation in the rattlesnake was significantly attenuated by prior treatment with propranolol (Fig. 7), indicating that a considerable portion of this response is mediated by stimulation of -adrenergic receptors. This would indicate that BK causes release of catecholamines from adrenergic presynaptic neurons or the adrenal medulla. Not having measured plasma concentrations of epinephrine and norepinephrine in response to BK, we cannot discern between these two possibilities. However, in mammals and pythons, injection of BK leads to a rise in circulating catecholamines, which is released from sympathetic neurons (27, 28).
Given that part of the BK-induced vasodilatation persisted after treatment with propranolol, other mechanisms seem to be involved in this response. BK failed to induce a significant vasodilatation after injection of L-NAME, implying that increased synthesis of NO contributes to the cardiovascular response to BK. The mechanism by which BK dilates the systemic vasculature differs among the different groups of vertebrates. As in the rattlesnake, the systemic vasodilatation caused by BK in the python (P. regius) was abolished after treatment with the -adrenergic receptor antagonist sotalol, indicating that this response is mediated by stimulation of -adrenergic receptors. However, in mammals, the vasodilator effects of BK have been attributed to both the activation of prostaglandins and through NO release (11, 20).
Having injected epinephrine and propranolol to unravel the mechanisms behind the action of BK, our study also yields some information regarding adrenergic tone in rattlesnakes. Injection of epinephrine caused a significant systemic vasoconstriction that persisted after treatment with propranolol, indicating that this response is mediated by stimulation of 
-adrenergic receptors. Furthermore, injection of propranolol caused a decrease in Gsys, indicating a basal -adrenergic tone on the systemic vasculature. Similar responses have been reported in other reptiles (13, 16, 21, 23, 28).
The tachycardia caused by BK (Fig. 7) was abolished by propranolol in rattlesnakes, indicating that it is caused by stimulation of -adrenergic receptors. This is also the case in python, where the tachycardia was virtually abolished after treatment with sotalol (28) and where circulating catecholamines increased in response to BK (28). Similarly, the tachycardia associated with the secondary response to BK in mammals has been ascribed to presynaptic release of catecholamines (27).
This is the first study to address the role of BK in the pulmonary circulation in a nonmammalian vertebrate. The pulmonary circulation of rattlesnakes was relatively unaffected by BK. In conjunction with this, several other vasoactive substances appear to have small effects on the pulmonary circulation in reptiles. In pythons, injection of neuropeptide 
causes a dose-dependent vasodilatation in the systemic circulation, while it has little effect on the pulmonary vasculature (Skovgaard, Galli, Taylor, Conlon, and Wang, unpublished observations). Similarly, in the American alligator, injection of endothelin-3 caused a decrease in systemic resistance, while the pulmonary circulation was unaffected (24). In Crotalus, NO contributes to the resting tone of the systemic circulation, and infusion of the NO donor sodium nitroprusside caused a systemic vasodilatation, but there were no effects on the pulmonary circulation (Galli, Skovgaard, Abe, Taylor, Conlon, and Wang, unpublished observations). Finally, in most reptiles, sympathetic regulation seems to be more pronounced in the systemic circulation compared with the pulmonary circulation. In the turtle, T. scripta, injection of catecholamines generally causes a small vasodilatation in the lungs, whereas the systemic effects are marked (1, 3, 5, 18, 23). Isolated pulmonary arteries from the snake, Elaphe obsolete, and the lizard, Trachydosaurus rugosus, exhibit a -adrenergically mediated pulmonary relaxation, while adrenergic stimulation failed to invoke a response in the pulmonary artery of the tortoise, Chelodina longicollis (1). However, in the rattlesnake, epinephrine caused a marked vasodilation in the pulmonary circulation that was abolished with propranolol (Table 2).
Perspectives
In both mammals and those species of reptiles studied, BK consistently causes a primary dose-dependent systemic vasodilatation. The sensitivity of the cardiovascular system of the python and the rattlesnake to BK [threshold 30 and 100 nmol/kg, respectively (28)] implies that this peptide may have a physiologically relevant role for cardiovascular regulation. In mammals, circulating levels of BK rise in response to tissue injury, stimulating the release of prostaglandins and the synthesis of NO, which alter distribution of blood flow to the damaged tissue area (11, 20, 25). In snakes, BK seems to exert its vasodilatory effects through stimulation of adrenergic release from sympathetic neurons. Nevertheless, the resulting cardiovascular effects are similar. Thus we would speculate that although the mechanisms through which BK acts may differ somewhat between mammals and snakes, the resulting cardiovascular actions and its functional significance in the response to tissue injury may be the same.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  N. Skovgaard, A. S. Abe, D. V. Andrade, and T. Wang Hypoxic pulmonary vasoconstriction in reptiles: a comparative study of four species with different lung structures and pulmonary blood pressures Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1280 - R1288. [Abstract] [Full Text] [PDF]
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