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Hemodynamic Responses of Broiler Pulmonary Vasculature to Intravenously Infused Serotonin
PHYSIOLOGY AND REPRODUCTION Hemodynamic Responses of Broiler Pulmonary Vasculature to Intravenously Infused Serotonin M. E. Chapman1 and R. F. Wideman, Jr. Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 over the course of a 10-min infusion period. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial pressure returned toward pre-infusion base-line values. Pulmonary hypertensive responses were associated with increased pulmonary vascular resistance (pulmonary vasoconstriction). The peak pulmonary arterial pressure attainable was inadequate to propel the normal cardiac output through the elevated pulmonary vascular resistance. Consequently, the impeded venous return to the left ventricle caused dependent reductions in stroke volume, cardiac output, and mean systemic arterial pressure. Reductions in cardiac output were associated with reductions in stroke volume but not heart rate. Any factor that reduces the pulmonary vascular capacity or increases the pulmonary vascular resistance theoretically can increase the incidence of PHS. The present study provides direct evidence that serotonin can trigger pulmonary vasoconstriction and pulmonary hypertension in broilers.
(Key words: pulmonary hypertension, vasoconstriction, broiler, serotonin, ascites) 2002 Poultry Science 81:231–238
that is synthesized from the essential amino acid tryptophan, actively accumulated by mammalian platelets and avian thrombocytes, and released into the plasma during platelet or thrombocyte aggregation (Meyer and Sturkie, 1974; Cox, 1985; Lacoste-Eleaume et al., 1994). 5HT has been implicated in the mechanisms responsible for pulmonary hypertension in several human and animal studies (Seiler et al., 1974; Douglas et al., 1981; Brenot et al., 1993; Abenhaim et al., 1996); however, the capacity of 5HT to trigger pulmonary hypertension in broilers has not previously been evaluated. Human patients with primary pulmonary hypertension (PPH) have elevated plasma 5HT concentrations coupled with reduced 5HT concentrations in their platelets. These PPH patients continue to exhibit elevated plasma 5HT levels after heart-lung transplantation, indicating that the high 5HT levels are not secondary to the vascular
INTRODUCTION Pulmonary hypertension syndrome (PHS) in broilers is associated with right ventricular hypertrophy and an elevated blood pressure within the pulmonary circulation (Cueva et al., 1974). PHS initiates the sequential development of hypoxemia, right-sided congestive heart failure, central venous congestion, cirrhosis of the liver, and accumulation of ascitic fluid into the abdominal cavity. Any factor that increases the cardiac output, that reduces the pulmonary vascular capacity, or that triggers pulmonary vasoconstriction can contribute to the pathogenesis of PHS in broilers (Wideman, 2000). Serotonin (5-hydoxytryptamine, 5HT) is a potent pulmonary vasoconstrictor
2002 Poultry Science Association, Inc. Received for publication March 19, 2001. Accepted for publication September 27, 2001. 1 To whom correspondence should be addressed: mchapman@ uark.edu.
Abbreviation Key: 5-HT = 5-hydoxytryptamine; PHS = pulmonary hypertension syndrome; PPH = primary pulmonary hypertension.
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ABSTRACT Serotonin is a potent pulmonary vasoconstrictor actively accumulated by mammalian platelets and avian thrombocytes and released into the plasma during platelet or thrombocyte aggregation. Serotonin has been implicated in the mechanisms responsible for pulmonary hypertension in several human and animal studies. However, the role of serotonin in pulmonary hypertension syndrome (PHS, ascites) in broilers previously had not been evaluated. In the present study we evaluated the pulmonary hemodynamic responses of broilers to intravenous infusions of serotonin dissolved in 2.5% (wt/vol) mannitol solution (carrier vehicle). Carrier vehicle infusion alone had no influence on any of the hemodynamic variables. Serotonin infusion triggered rapid increases in pulmonary arterial pressure to approximately 50% above pre-infusion baseline values, accompanied by decreases in mean systemic arterial pressure and cardiac output. The peak pulmonary arterial pressure response occurred within approximately 70 s after the start of serotonin infusion and remained elevated above baseline values
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MATERIALS AND METHODS Male chicks were hatched on August 24, 2000, at the Hubbard ISA hatchery.2 The chicks were wing-banded
2
Hubbard ISA, Hot Springs, AR 71902. Sigma Chemical Co., St. Louis, MO 63178-9916. Henry Schein, Inc., Melville, NY 11747. 5 Transonic Systems, Inc., Ithaca, NY 14850. 6 Konigsberg Instruments, Inc., Pasadena, CA 91107-3294. 7 World Precision Instruments, Sarasota, FL 34230. 8 Biopac Systems, Inc., Goleta, CA 93117. 3 4
and shipped on the day of hatch (Day 1) to the poultry environmental research laboratory at the University of Arkansas. They were placed on fresh wood shavings in environmental chambers (8 m2 floor space) and were brooded at 33 C from Days 1 to 5, 29 C from Days 6 to 10, and 27 C from Days 11 to 17. Thereafter, the broilers were maintained at 21 C until the experiment was terminated. The photoperiod was 24 h of light from Days 1 to 5 and 23 h of light/1 h of darkness thereafter. Water was provided ad libitum via bell-type waterers. A cornsoybean meal starter ration (22.7%CP, 3,059 kcal ME/kg, 1.5% arginine, and 1.43% lysine) was provided ad libitum and had been formulated to meet or exceed the minimum NRC (1984) standards for all ingredients. The diet was provided as crumbles during Weeks 1 and 2 and as pellets thereafter.
Experiment 1 Ten birds (2,147 ± 71 g, mean ± SEM) were anesthetized to a light surgical plane with intramuscular injections of allobarbitol (5,5-diallylbarbituric acid3 3.0 mL, 25 mg/ml) and ketamine HCl4 (1.0 mL, 100 mg/mL). The birds were placed on a heated surgical board (30 C) and restrained in dorsal recumbancy. All blood pressure readings were made with the transducer at the level of the thoracic inlet. After 2% (wt/vol) lidocaine HCl had been administered intracutaneously as a local anesthetic, an incision was made to open the thoracic inlet, and the left and right pulmonary arteries were exposed. A transonic ultrasonic flow probe was positioned on the right pulmonary artery, and the probe was connected to a Transonic T2065 blood flow meter to confirm signal acquisition. A 27-ga × 1-in. needle, bent to a 90°-angle midway along its length, was scored and snapped off adjacent to the hub. The resulting blunt end was pressure fit into a 30-cm length of Silastic6 tubing (0.012 in. I.D., 0.037 in. O.D.) filled with 0.8% (wt/ vol) sodium chloride containing 200 IU heparin/mL. The needle was inserted into the left pulmonary artery, and the thoracic inlet was sealed with stainless steel wound clips. The Silastic tubing was attached to a blood pressure transducer interfaced through a Transbridge preamplifier7 to a Biopac MP 10 data acquisition system using AcqKnowledge8 software. The left basilica vein was cannulated with PE-50 polyethylene tubing for i.v. infusions. The left brachial artery was cannulated with PE-50 polyethylene tubing filled with heparinized saline and was attached to a blood pressure transducer for monitoring of systemic arterial pressure. After surgical preparations were complete and 5 min stabilization had elapsed, control data were recorded for 5 min. Mannitol (2.5% wt/vol, 25 g mannitol/L of water) was infused through the PE-50 tubing in the left basilica vein at 0.1 mL/min per kg per BW to hydrate the bird (Wideman and Gregg, 1988) and act as a volume control and carrier vehicle. Serotonin (5-HT maleate salt,3 0.1 mg in 20 mL 2.5% mannitol) was infused at 0.1 mL/min per kg per BW through the PE-50 tubing in the left basilica vein for 10 min, after which 2.5% (wt/vol) mannitol infu-
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changes present in pulmonary hypertension (Herve et al., 1995). Dexfenfluramine is an appetite-suppressant drug that causes 5HT to be released from platelets and inhibits its re-uptake (Weir et al., 1996). Evaluations of 37 patients with PPH who had used dexfenfluramine suggested a direct involvement of 5HT in the onset of PPH (Douglas et al., 1981, Brenot et al., 1993, Abenhaim et al., 1996). In addition, Seiler et al. (1974) showed that the appetitesuppressant drugs aminorex and chlorphentermine impaired uptake of 5HT by isolated perfused rat lungs, resulting in elevated plasma 5HT levels and persistent pulmonary vasoconstriction. These results suggest an intrinsic abnormality of platelet 5HT uptake and release may contribute to the etiology of pulmonary hypertension in mammals. Many cells are capable of synthesizing 5HT; however, the majority of circulating 5HT is produced by endocrine cells in the gastrointestinal tract. 5HT is released into the blood from the gut and enters platelets via membrane transporter proteins (2A receptors) and is stored inside the platelets in the form of dense granules. Avian thrombocytes contain numerous dense granules consisting of concentrated 5HT (Kuruma et al. 1970). The importance of platelets in controlling the plasma concentration of 5HT is illustrated by platelet delta storage pool disease. In the inherited condition, there is a deficiency in the number and content of dense granules within platelets. In one patient who also developed PPH, the plasma concentration of 5HT increased 15-fold (Herve et al., 1990). Moreover, fawn hooded rats, a species that has a tendency to develop pulmonary hypertension, have a similar inherited platelet disorder (Sato et al., 1992). 5HT release is responsible for platelet secretion, aggregation, and formation of clots. As platelets aggregate, they release several physiologically active substances, including serotonin, which cause proliferation of pulmonary vascular smooth muscle and stimulate vasoconstriction, thereby reducing the blood flow at the site of injury (McGoon and Vanhoutte, 1984; Lee et al., 1994; Pitt et al., 1994; Fanburg and Lee, 1997). In domesticated avian species, biogenic amines are known to cause pulmonary vasoconstriction (Wideman, 1999), and thrombocyte aggregation has been observed in response to serotonin in vitro (Belamarich et al., 1968). In the present study, hemodynamic responses of the broiler pulmonary vasculature to intravenously infused 5HT were evaluated.
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sion was resumed for a further 5 min. After a satisfactory recording was obtained, the birds were euthanized with 10 mL i.v. of 0.1 M KCl.
Experiment 2
Data Analysis The primary channels recorded by the Biopac MP 100 data acquisition system included systemic arterial pressure (mm Hg), pulmonary arterial pressure (mm Hg), and blood flow through the right pulmonary artery (mL/ min). Heart rate (beats/min) was obtained by counting systolic peaks over time in the pulmonary arterial pressure recording. These primary data were averaged electronically during representative sample intervals at the start of data collection (start), at 5-min intervals throughout the control period (C, C5), within 30 s after infusing 5HT (SI), at 5-min intervals after 5HT infusion (SI5, SI10), within 30 s of ending the 5HT infusion (EI), and at the end of the experiment (EI5). The protocol used for data averaging accommodates the influences of pulse pressure
and respiratory cycles on pulmonary and systemic arterial pressures. (Wideman et al., 1996). The primary data were used to calculate cardiac output, stroke volume, pulmonary vascular resistance, and total peripheral resistance. Based on the assumption that cardiac output (mL/min) normally is divided equally between the lungs, cardiac output was calculated as 2 × blood flow. The cardiac output is the product of heart rate × stroke volume (mL/ beat), consequently stroke volume was calculated as cardiac output divided by heart rate. Assuming the pressure gradients across the pulmonary and systemic circulations are essentially equal to pulmonary arterial pressure and systemic arterial pressure, respectively (Wideman et al., 1996, 1998), then the relationships between pressure gradients, flow rates, and resistances are summarized by the respective equations. Pulmonary arterial pressure = cardiac output × pulmonary vascular resistance, and mean systemic arterial pressure = cardiac output × total peripheral resistance. Thus, pulmonary vascular resistance was calculated in relative resistance units as pulmonary arterial pressure (mm Hg) divided by cardiac output (mL/min), and total peripheral resistance was calculated in relative resistance units as mean systemic arterial pressure (mm Hg) divided by cardiac output (mL/min) as described by Besch and Kadono (1978), Sturkie (1986), and Wideman et al. (1996, 1998). Due to the direct proportionality between cardiac output and body weight (Wideman, 1999), all data derived from blood flow measurements were normalized for body weight on an individual bird basis, prior to calculating group means. The data were analyzed using ANOVA or ANOVA on ranks using the Student-Newman-Keuls method for the separation of treatment means (Jandel Scientific, 1994).
RESULTS Mannitol infusion (Figures 1 to 6) and general anesthesia (Figures 2 and 6) had no influence on any of the hemodynamic variables. During pilot studies conducted to survey effective i.v. infusion dosages for 5HT, infusing 1.0 to 0.5 mg 5HT in 20mL of 2.5% mannitol triggered massive pulmonary vasoconstriction leading to an immediate (within 30 s) >90% reduction in cardiac output and death (not shown). Thereafter, 0.1 mg 5HT in 20 mL of 2.5% (wt/vol) mannitol was established as efficacious intermediate dosage that permitted 100% survival and postinfusion recovery toward control values by the experimental animals. Typical hemodynamic responses of individual broilers to i.v. infusions of 5HT are shown in Figures 1 and 2. 5HT infusion triggered rapid increases in pulmonary arterial pressure accompanied by decreases in mean systemic arterial pressure and cardiac output. 5HT infusions increased pulmonary arterial pressure by approximately 50% above preinfusion baseline values (Figure 3), and the peak pulmonary arterial pressure response occurred approximately 70 s after the start of 5HT infusion. These pulmonary hypertensive responses were associated with increases in pulmonary vascular resistance (Figure 3). The pulmonary hypertensive response
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To evaluate the influence of general anesthesia and surgical preparation on the pulmonary hypertensive responses to serotonin, pulmonary arterial pressures were measured using unanaesthetized broilers. Nine unanaesthetized broilers were restrained on a surgical board, heated to 30 C, in dorsal recumbancy. The feathers on the ventral surface of the wing between the elbow and shoulder joint were plucked. An incision was made over the basilica vein after 2% (wt/vol) lidocaine HCl had been administered intracutaneously as a local anesthetic. A Silastic catheter (0.012 in. I.D., 0.037 in. O.D.) filled with heparinized saline was inserted into the basilica vein. The distal end of the catheter was attached to a blood pressure transducer interfaced through a Transbridge preamplifier to a Biopac MP 100 data acquisition system using AcqKnowledge software. Pressure recordings were begun and the proximal end of the catheter was advanced through the right atrium and ventricle into a pulmonary artery while monitoring the characteristic pulse pressures to identify the location (Owen et al., 1995b; Chapman and Wideman, 2001). The catheter was then slowly advanced until it was positioned in the pulmonary artery. Prior to recording, the system was calibrated for accuracy using a mercury manometer. After surgical preparations were complete and a 5-min stabilization had elapsed, control data were recorded for 5 min. Mannitol (2.5%, 0.1 mL/min per kg per BW) was infused through the PE-50 tubing in the left basilica vein to hydrate the bird (Wideman and Gregg, 1988) and act as a volume control and carrier vehicle. Serotonin (0.1 mg in 20 mL 2.5% mannitol) was infused at 0.1 mL/min per kg per BW through the PE-50 tubing in the left basilica vein for 10 min, after which 2.5% (wt/vol) mannitol infusion was resumed for a further 5 min. After a satisfactory recording was obtained, the birds were euthanized with 10 mL i.v. of 0.1 M KCl.
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FIGURE 2. Physiograph recording from an individual unanaesthetized male broiler in Experiment 2, showing continuous values for pulmonary arterial pressure (PAP) during a 5-min control period, after a 5-min infusion of 2.5% mannitol as a volume control, after a 10-min infusion of serotonin (0.1 mg in 20 mL 2.5% mannitol), followed by a further 5-min infusion of 2.5% mannitol.
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FIGURE 1. Physiograph recording from an individual male broiler in Experiment 1, showing continuous values for mean systemic arterial pressure (MAP), pulmonary arterial pressure (PAP), and blood flow through the right pulmonary artery (Flow) during a 5-min control period, after a 5-min infusion of 2.5% mannitol as a volume control, after a 10-min infusion of serotonin (0.1 mg in 20 mL 2.5% mannitol), followed by a further 5-min infusion of 2.5% mannitol.
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pressure returned toward pre-infusion base-line values. The pulmonary hypertensive responses of unanesthetized broilers were similar to those of anesthetized broilers (Figures 2 and 6).
DISCUSSION
FIGURE 3. Pulmonary arterial pressure (PAP, upper panel), pulmonary vascular resistance (PVR, middle panel), and blood flow through the right pulmonary artery (Flow, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-d) designate differences between means over time (P ≤ 0.05).
tended to decline as the 10-min infusion of 5HT progressed, but pulmonary arterial pressure remained elevated above baseline values throughout the interval of 5HT infusion. Blood flow through the pulmonary artery was depressed throughout the period of 5HT infusion (Figure 3). Contemporaneous reductions in cardiac output were associated with reductions in stroke volume but not with a reduction of heart rate (Figure 4). Mean systemic arterial pressure dropped below pre-infusion baseline levels during the pulmonary hypertensive responses to 5HT (Figure 5). Mean systemic arterial pressure and cardiac output responses also tended to be attenuated over the 10-min course of 5HT infusion but remained lower than the pre- or postinfusion baseline values. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial
FIGURE 4. Cardiac output (CO, upper panel), stroke volume (SV, middle panel), and heart rate (HR, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P ≤ 0.05); ns = not significant (P > 0.05).
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Any factor that reduces the pulmonary vascular capacity or increases the pulmonary vascular resistance theoretically can increase the incidence of PHS (Wideman and Bottje, 1993; Wideman, 2000). Serotonin infusion triggered rapid increases in pulmonary arterial pressure to approximately 50% above pre-infusion baseline values, accompanied by decreases in mean systemic arterial pressure and cardiac output, whereas carrier vehicle infusion alone had no influence on any of the hemodynamic variables. Pulmonary hypertensive responses were associated with increased pulmonary vascular resistance (pulmo-
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FIGURE 6. Pulmonary arterial pressure (PAP) for unanaesthetized male broilers (mean ± SEM, n = 9) in Experiment 2, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and at 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P ≤ 0.05).
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FIGURE 5. Mean systemic arterial pressure (MAP, upper panel), and total peripheral resistance (TPR, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5 min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and at 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P > 0.05); ns = not significant (P > 0.05).
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ACKNOWLEDGMENTS This research was supported by a US-Israel Binational Agricultural Research and Development grant (BARD US-2736-96) and by a grant from Hubbard ISA, Walpole, NH 03608.
REFERENCES Abenhaim, L., Y. Moride, F. Brenot, S. Rich, J. Benichou, X. Kurz, T. Higenbottam, C. Oakley, E. Wouters, M. Aubier, G. Simonneau, and B. Begaud, 1996. Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N. Engl. J. Med. 335:609–616. Belamarich, F. A., D. Sherpo, and M. Kein, 1968. ADP is not involved in thrombin-induced aggregation of thrombocytes of a non-mammalian vertebrate. Nature 220:509–510. Besch, E. L., and H. Kadono, 1978. Cardiopulmonary responses to acute hypoxia in domestic fowl. Pages 71–78 in: Respiratory Function in Birds Adult and Embryonic. J. Piiper, ed. Springer-Verlag, New York, NY. Brenot, F., P. Herve, P. Petitpretz, F. Parent, P. Duroux, and G. Simmoneu, 1993. Primary pulmonary hypertension and fenfluramine use. Br. Heart J. 70:537–541. Chapman, M. E., and R. F. Wideman, Jr., 2001. Pulmonary wedge pressures confirm pulmonary hypertension in broilers is initiated by an excessive pulmonary arterial (Precapillary) resistance. Poultry Sci. 80:468–473. Cox, C. P., 1985. Activation of washed chicken thrombocytes by 1-0-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (platelet activating factor). Comp. Biochem. Physiol. 82A:145–151. Cueva, S., H. Sillau, A. Valenzuela, and H. Ploog, 1974. High altitude induced pulmonary hypertension and right ventricular failure in broiler chickens. Res. Vet. Sci. 16:370–374. Dekker, G. A., 1995. The pharmacological prevention of preeclampsia. Baillieres Clin. Obstet. Gynaecol. 9:509–528. Douglas, J. G., J. F. Munro, A. H. Kitchin, A. L. Muir, and A. T. Proudfoot, 1981. Pulmonary hypertension and fenfluramine. BMJ 283:881–883. Fanburg, B. L., S. L. Lee, 1997. A new role for an old molecule: Serotonin as a mitogen. Am. J. Physiol. 272:L795–806. Heffner, J. E., and J. E. Repine, 1997. Platelets. Pages 947–959 in: The Lung: Scientific Foundations. 2nd ed. R. G. Crystal, J. B. West, P. J. Barnes, and E. R. Weibel, ed. LippincottRaven, Philadelphia, PA. Herve, P., L. Drouet, C. Dosquet, J. M. Launay, B. Rain, G. Simonneau, J. Caen, and P. Duroux, 1990. Primary pulmonary hypertension in a patient with a familial platelet storage pool disease: Role of serotonin. Am. J. Med. 89:117–120. Herve´, P., J. M. Launay, M. L. Scrobohaci, F. Brenot, G. Simonneau, P. Petitpretz, P. Poubeau, J. Cerrina, P. Duroux, and L. Drouet, 1995. Increased plasma serotonin in primary pulmonary hypertension. Am. J. Med. 99:249–254. Jandel Scientific, 1994. SigmaStat威 Statistical Software User’s Manual. Jandel Scientific Software, San Rafael, CA. Kuruma, I., T. Okada, K. Kataoka, and M. Sorimachi, 1970. Ultrastructural observation of 5-hydroxytryptamine-storing granules in the domestic fowl thrombocytes. Z. Zellforsch. 108:268–281. Lacoste-Eleaume, A. S., C. Bleux, P. Quere, F. Coudert, C. Corbel, and C. Kanellopoulos-Langevin, 1994. Biochemical and functional characterization of an avian homolog of the integrin GPIIb-IIIa present on chicken thrombocytes. Exp. Cell Res. 213:198–209. Lee, S. L., W. W. Weng, J. J. Lanzillo, and B. L. Fanburg, 1994. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am. J. Physiol. 266:146–152. Maxwell, M. H., T. T. Dolan, and H. C. W. Mbuga, 1989. An ultrastructural study of an ascitic syndrome in young broilers reared at high altitude. Avian Pathol. 18:481–494. Maxwell, M. H., G. W. Robertson, and S. Spence, 1986. Studies on an ascitic syndrome in young broilers 2. Ultrastructure. Avian Pathol. 15:525–538.
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nary vasoconstriction). The peak pulmonary arterial pressure attainable was inadequate to propel the normal cardiac output through the elevated pulmonary vascular resistance. Consequently, the impeded venous return to the left ventricle caused dependant reductions in stroke volume, cardiac output, and mean systemic arterial pressure. Reductions in cardiac output were not associated with reductions in heart rate. The systemic hypotension that coincides with increased pulmonary vascular resistance reflects the inability of the relatively weak right ventricle to sustain a normal cardiac output. The peak pulmonary arterial pressure response occurred within approximately 70 s after the start of 5HT infusion, tended to decline over the course of a 10-min infusion period, but remained elevated above baseline values. The pulmonary hypertensive response to 5HT might have been partially attenuated by flow-dependent vasodilation mediated by the pulmonary vascular endothelium. Utilization of plasma L-arginine as a substrate for the production of the vasodilator nitric oxide might have relaxed the pre-existing tone of the pulmonary vessels, thereby allowing increased flow through the pulmonary vasculature at a lower pressure. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial pressure returned toward pre-infusion base-line values. The present study provides direct evidence that 5HT can trigger pulmonary vasoconstriction and pulmonary hypertension in broilers. The significance of this observation relates to the proposal that pulmonary hypertension may be triggered in susceptible broilers by poor air quality and respiratory disease. Pulmonary endothelial-cell damage has been associated with platelet activation in humans, resulting in increased release of 5HT (Dekker, 1995). Furthermore, elevated plasma concentrations of 5HT have been implicated in the pulmonary hypertension associated with acute respiratory disease syndrome in humans (Heffner and Repine, 1997). Lungs of broilers developing ascites contain accumulations of mast cells, macrophages, and inflammatory foci (Maxwell et al., 1986, 1989; Owen et al., 1995a). In addition to antigens entering the lungs through inspired air, venous blood returning to the right ventricle of the heart also creates a continuous pulmonary exposure to blood-borne particles. In one study, bolus intravenous injections of bacterial endotoxin were shown to trigger pulmonary vasoconstriction and pulmonary hypertension in broiler chickens (Wideman et. al., 2001). This process can be profoundly damaging to pulmonary hemodynamics and gas exchange and may contribute to the pathogenesis of PHS.
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CHAPMAN AND WIDEMAN, JR. Weir, E. K., H. L. Reeve, J. M. Huang, E. Michelakis, D. P. Nelson, V. Hampl, and S. L. Archer, 1996. Anorexic agents aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in rat pulmonary vascular smooth muscle and cause pulmonary vasoconstriction. Circulation 94:2216–2220. Wideman, R. F., 1999. Cardiac Output in four-, five-, and sixweek-old broilers, and hemodynamic responses to intravenous injections of epinephrine. Poultry Sci. 78:392–403. Wideman, R. F., 2000. Cardio-pulmonary hemodynamics and ascites in broiler chickens. Poult. Avian Biol. Rev. 11:1–23. Wideman, R. F., and W. G. Bottje, 1993. Current understanding of the ascites syndrome and future research directions. Pages 1–20 in: Nutritional and Technical Symposium Proceedings. Novus International Inc., St. Louis, MO. Wideman, Jr., R. F., G. F. Erf, and M. E. Chapman, 2001. Intravenous endotoxin triggers pulmonary vasoconstriction and pulmonary hypertension in broiler chickens. Poultry Sci. 80:647–655. Wideman, R. F., and C. M. Gregg, 1988. Model for evaluating avian renal hemodynamics and glomerular filtration rate autoregulation. Am. J. Physiol. 254:R925–R932. Wideman, R. F., Y. K. Kirby, M. F. Forman, N. Marson, R. W. McNew, and R. L. Owen, 1998. The infusion rate dependant influence of metabolic acidosis on pulmonary vascular resistance in broilers. Poultry Sci. 77:309–321. Wideman, R. F., Y. K. Kirby, C. D. Tacket, N. E. Marson, and R. W. McNew, 1996. Cardio-pulmonary function during acute unilateral occlusion of the pulmonary artery in broilers fed diets containing normal or high levels of arginine-HCl. Poultry Sci. 75:1587–1602.
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McGoon, M. D., and P. M. Vanhoutte, 1984. Aggregating platelets contract isolated canine pulmonary arteries by releasing 5-hydroxytryptamine. J. Clin. Invest. 74:828–833. Meyer, D. C., and P. D. Sturkie, 1974. Distribution of serotonin among blood cells of the domestic fowl. Proc. Soc. Exp. Biol. Med. 147:382–386. National Research Council, 1984. Nutrition Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Owen, R. L., R. F. Wideman, and B. S. Cowen, 1995a. Morphometric and histologic changes in the pulmonary system of broilers raised at simulated high altitude. Avian Pathol. 24:293–302. Owen, R. L., R. F. Wideman, and B. S. Cowen, 1995b. Changes in pulmonary arterial and femoral arterial blood pressure upon acute exposure to hypobaric hypoxia in broiler chickens. Poultry Sci. 74:708–715. Pitt, B. R., W. Weng, A. R. Steve, R. D. Blakely, I. Reynolds, and P. Davies, 1994. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am. J. Physiol. 266:L178–L186. Sato, K., S. Webb, A. Tucker, M. Rabinovitch, R. F. O’brien, I. F. McMurty, and T. J. Stelzner, 1992. Factors influencing the idiopathic development of pulmonary hypertension in the fawn hooded rat. Am. Rev. Respir. Dis. 145:793–797. Seiler, K. U., O. Wassermann, and H. Wensky, 1974. On the role of serotonin in the pathogenesis of pulmonary hypertension induced by anorectic drugs, an experimental study in the isolated perfused rat lung. Clin. Exp. Pharmacol. Physiol. 1:463–471. Sturkie, P. D., 1986. Body fluids: blood. Pages 102–109 in: Avian Physiology. 4th ed. P. J. Sturkie, ed. Springer-Verlag, New York, NY.
(Key words: pulmonary hypertension, vasoconstriction, broiler, serotonin, ascites) 2002 Poultry Science 81:231–238
that is synthesized from the essential amino acid tryptophan, actively accumulated by mammalian platelets and avian thrombocytes, and released into the plasma during platelet or thrombocyte aggregation (Meyer and Sturkie, 1974; Cox, 1985; Lacoste-Eleaume et al., 1994). 5HT has been implicated in the mechanisms responsible for pulmonary hypertension in several human and animal studies (Seiler et al., 1974; Douglas et al., 1981; Brenot et al., 1993; Abenhaim et al., 1996); however, the capacity of 5HT to trigger pulmonary hypertension in broilers has not previously been evaluated. Human patients with primary pulmonary hypertension (PPH) have elevated plasma 5HT concentrations coupled with reduced 5HT concentrations in their platelets. These PPH patients continue to exhibit elevated plasma 5HT levels after heart-lung transplantation, indicating that the high 5HT levels are not secondary to the vascular
INTRODUCTION Pulmonary hypertension syndrome (PHS) in broilers is associated with right ventricular hypertrophy and an elevated blood pressure within the pulmonary circulation (Cueva et al., 1974). PHS initiates the sequential development of hypoxemia, right-sided congestive heart failure, central venous congestion, cirrhosis of the liver, and accumulation of ascitic fluid into the abdominal cavity. Any factor that increases the cardiac output, that reduces the pulmonary vascular capacity, or that triggers pulmonary vasoconstriction can contribute to the pathogenesis of PHS in broilers (Wideman, 2000). Serotonin (5-hydoxytryptamine, 5HT) is a potent pulmonary vasoconstrictor
2002 Poultry Science Association, Inc. Received for publication March 19, 2001. Accepted for publication September 27, 2001. 1 To whom correspondence should be addressed: mchapman@ uark.edu.
Abbreviation Key: 5-HT = 5-hydoxytryptamine; PHS = pulmonary hypertension syndrome; PPH = primary pulmonary hypertension.
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ABSTRACT Serotonin is a potent pulmonary vasoconstrictor actively accumulated by mammalian platelets and avian thrombocytes and released into the plasma during platelet or thrombocyte aggregation. Serotonin has been implicated in the mechanisms responsible for pulmonary hypertension in several human and animal studies. However, the role of serotonin in pulmonary hypertension syndrome (PHS, ascites) in broilers previously had not been evaluated. In the present study we evaluated the pulmonary hemodynamic responses of broilers to intravenous infusions of serotonin dissolved in 2.5% (wt/vol) mannitol solution (carrier vehicle). Carrier vehicle infusion alone had no influence on any of the hemodynamic variables. Serotonin infusion triggered rapid increases in pulmonary arterial pressure to approximately 50% above pre-infusion baseline values, accompanied by decreases in mean systemic arterial pressure and cardiac output. The peak pulmonary arterial pressure response occurred within approximately 70 s after the start of serotonin infusion and remained elevated above baseline values
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MATERIALS AND METHODS Male chicks were hatched on August 24, 2000, at the Hubbard ISA hatchery.2 The chicks were wing-banded
2
Hubbard ISA, Hot Springs, AR 71902. Sigma Chemical Co., St. Louis, MO 63178-9916. Henry Schein, Inc., Melville, NY 11747. 5 Transonic Systems, Inc., Ithaca, NY 14850. 6 Konigsberg Instruments, Inc., Pasadena, CA 91107-3294. 7 World Precision Instruments, Sarasota, FL 34230. 8 Biopac Systems, Inc., Goleta, CA 93117. 3 4
and shipped on the day of hatch (Day 1) to the poultry environmental research laboratory at the University of Arkansas. They were placed on fresh wood shavings in environmental chambers (8 m2 floor space) and were brooded at 33 C from Days 1 to 5, 29 C from Days 6 to 10, and 27 C from Days 11 to 17. Thereafter, the broilers were maintained at 21 C until the experiment was terminated. The photoperiod was 24 h of light from Days 1 to 5 and 23 h of light/1 h of darkness thereafter. Water was provided ad libitum via bell-type waterers. A cornsoybean meal starter ration (22.7%CP, 3,059 kcal ME/kg, 1.5% arginine, and 1.43% lysine) was provided ad libitum and had been formulated to meet or exceed the minimum NRC (1984) standards for all ingredients. The diet was provided as crumbles during Weeks 1 and 2 and as pellets thereafter.
Experiment 1 Ten birds (2,147 ± 71 g, mean ± SEM) were anesthetized to a light surgical plane with intramuscular injections of allobarbitol (5,5-diallylbarbituric acid3 3.0 mL, 25 mg/ml) and ketamine HCl4 (1.0 mL, 100 mg/mL). The birds were placed on a heated surgical board (30 C) and restrained in dorsal recumbancy. All blood pressure readings were made with the transducer at the level of the thoracic inlet. After 2% (wt/vol) lidocaine HCl had been administered intracutaneously as a local anesthetic, an incision was made to open the thoracic inlet, and the left and right pulmonary arteries were exposed. A transonic ultrasonic flow probe was positioned on the right pulmonary artery, and the probe was connected to a Transonic T2065 blood flow meter to confirm signal acquisition. A 27-ga × 1-in. needle, bent to a 90°-angle midway along its length, was scored and snapped off adjacent to the hub. The resulting blunt end was pressure fit into a 30-cm length of Silastic6 tubing (0.012 in. I.D., 0.037 in. O.D.) filled with 0.8% (wt/ vol) sodium chloride containing 200 IU heparin/mL. The needle was inserted into the left pulmonary artery, and the thoracic inlet was sealed with stainless steel wound clips. The Silastic tubing was attached to a blood pressure transducer interfaced through a Transbridge preamplifier7 to a Biopac MP 10 data acquisition system using AcqKnowledge8 software. The left basilica vein was cannulated with PE-50 polyethylene tubing for i.v. infusions. The left brachial artery was cannulated with PE-50 polyethylene tubing filled with heparinized saline and was attached to a blood pressure transducer for monitoring of systemic arterial pressure. After surgical preparations were complete and 5 min stabilization had elapsed, control data were recorded for 5 min. Mannitol (2.5% wt/vol, 25 g mannitol/L of water) was infused through the PE-50 tubing in the left basilica vein at 0.1 mL/min per kg per BW to hydrate the bird (Wideman and Gregg, 1988) and act as a volume control and carrier vehicle. Serotonin (5-HT maleate salt,3 0.1 mg in 20 mL 2.5% mannitol) was infused at 0.1 mL/min per kg per BW through the PE-50 tubing in the left basilica vein for 10 min, after which 2.5% (wt/vol) mannitol infu-
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changes present in pulmonary hypertension (Herve et al., 1995). Dexfenfluramine is an appetite-suppressant drug that causes 5HT to be released from platelets and inhibits its re-uptake (Weir et al., 1996). Evaluations of 37 patients with PPH who had used dexfenfluramine suggested a direct involvement of 5HT in the onset of PPH (Douglas et al., 1981, Brenot et al., 1993, Abenhaim et al., 1996). In addition, Seiler et al. (1974) showed that the appetitesuppressant drugs aminorex and chlorphentermine impaired uptake of 5HT by isolated perfused rat lungs, resulting in elevated plasma 5HT levels and persistent pulmonary vasoconstriction. These results suggest an intrinsic abnormality of platelet 5HT uptake and release may contribute to the etiology of pulmonary hypertension in mammals. Many cells are capable of synthesizing 5HT; however, the majority of circulating 5HT is produced by endocrine cells in the gastrointestinal tract. 5HT is released into the blood from the gut and enters platelets via membrane transporter proteins (2A receptors) and is stored inside the platelets in the form of dense granules. Avian thrombocytes contain numerous dense granules consisting of concentrated 5HT (Kuruma et al. 1970). The importance of platelets in controlling the plasma concentration of 5HT is illustrated by platelet delta storage pool disease. In the inherited condition, there is a deficiency in the number and content of dense granules within platelets. In one patient who also developed PPH, the plasma concentration of 5HT increased 15-fold (Herve et al., 1990). Moreover, fawn hooded rats, a species that has a tendency to develop pulmonary hypertension, have a similar inherited platelet disorder (Sato et al., 1992). 5HT release is responsible for platelet secretion, aggregation, and formation of clots. As platelets aggregate, they release several physiologically active substances, including serotonin, which cause proliferation of pulmonary vascular smooth muscle and stimulate vasoconstriction, thereby reducing the blood flow at the site of injury (McGoon and Vanhoutte, 1984; Lee et al., 1994; Pitt et al., 1994; Fanburg and Lee, 1997). In domesticated avian species, biogenic amines are known to cause pulmonary vasoconstriction (Wideman, 1999), and thrombocyte aggregation has been observed in response to serotonin in vitro (Belamarich et al., 1968). In the present study, hemodynamic responses of the broiler pulmonary vasculature to intravenously infused 5HT were evaluated.
EFFECTS OF SEROTONIN ON PULMONARY HYPERTENSION
sion was resumed for a further 5 min. After a satisfactory recording was obtained, the birds were euthanized with 10 mL i.v. of 0.1 M KCl.
Experiment 2
Data Analysis The primary channels recorded by the Biopac MP 100 data acquisition system included systemic arterial pressure (mm Hg), pulmonary arterial pressure (mm Hg), and blood flow through the right pulmonary artery (mL/ min). Heart rate (beats/min) was obtained by counting systolic peaks over time in the pulmonary arterial pressure recording. These primary data were averaged electronically during representative sample intervals at the start of data collection (start), at 5-min intervals throughout the control period (C, C5), within 30 s after infusing 5HT (SI), at 5-min intervals after 5HT infusion (SI5, SI10), within 30 s of ending the 5HT infusion (EI), and at the end of the experiment (EI5). The protocol used for data averaging accommodates the influences of pulse pressure
and respiratory cycles on pulmonary and systemic arterial pressures. (Wideman et al., 1996). The primary data were used to calculate cardiac output, stroke volume, pulmonary vascular resistance, and total peripheral resistance. Based on the assumption that cardiac output (mL/min) normally is divided equally between the lungs, cardiac output was calculated as 2 × blood flow. The cardiac output is the product of heart rate × stroke volume (mL/ beat), consequently stroke volume was calculated as cardiac output divided by heart rate. Assuming the pressure gradients across the pulmonary and systemic circulations are essentially equal to pulmonary arterial pressure and systemic arterial pressure, respectively (Wideman et al., 1996, 1998), then the relationships between pressure gradients, flow rates, and resistances are summarized by the respective equations. Pulmonary arterial pressure = cardiac output × pulmonary vascular resistance, and mean systemic arterial pressure = cardiac output × total peripheral resistance. Thus, pulmonary vascular resistance was calculated in relative resistance units as pulmonary arterial pressure (mm Hg) divided by cardiac output (mL/min), and total peripheral resistance was calculated in relative resistance units as mean systemic arterial pressure (mm Hg) divided by cardiac output (mL/min) as described by Besch and Kadono (1978), Sturkie (1986), and Wideman et al. (1996, 1998). Due to the direct proportionality between cardiac output and body weight (Wideman, 1999), all data derived from blood flow measurements were normalized for body weight on an individual bird basis, prior to calculating group means. The data were analyzed using ANOVA or ANOVA on ranks using the Student-Newman-Keuls method for the separation of treatment means (Jandel Scientific, 1994).
RESULTS Mannitol infusion (Figures 1 to 6) and general anesthesia (Figures 2 and 6) had no influence on any of the hemodynamic variables. During pilot studies conducted to survey effective i.v. infusion dosages for 5HT, infusing 1.0 to 0.5 mg 5HT in 20mL of 2.5% mannitol triggered massive pulmonary vasoconstriction leading to an immediate (within 30 s) >90% reduction in cardiac output and death (not shown). Thereafter, 0.1 mg 5HT in 20 mL of 2.5% (wt/vol) mannitol was established as efficacious intermediate dosage that permitted 100% survival and postinfusion recovery toward control values by the experimental animals. Typical hemodynamic responses of individual broilers to i.v. infusions of 5HT are shown in Figures 1 and 2. 5HT infusion triggered rapid increases in pulmonary arterial pressure accompanied by decreases in mean systemic arterial pressure and cardiac output. 5HT infusions increased pulmonary arterial pressure by approximately 50% above preinfusion baseline values (Figure 3), and the peak pulmonary arterial pressure response occurred approximately 70 s after the start of 5HT infusion. These pulmonary hypertensive responses were associated with increases in pulmonary vascular resistance (Figure 3). The pulmonary hypertensive response
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To evaluate the influence of general anesthesia and surgical preparation on the pulmonary hypertensive responses to serotonin, pulmonary arterial pressures were measured using unanaesthetized broilers. Nine unanaesthetized broilers were restrained on a surgical board, heated to 30 C, in dorsal recumbancy. The feathers on the ventral surface of the wing between the elbow and shoulder joint were plucked. An incision was made over the basilica vein after 2% (wt/vol) lidocaine HCl had been administered intracutaneously as a local anesthetic. A Silastic catheter (0.012 in. I.D., 0.037 in. O.D.) filled with heparinized saline was inserted into the basilica vein. The distal end of the catheter was attached to a blood pressure transducer interfaced through a Transbridge preamplifier to a Biopac MP 100 data acquisition system using AcqKnowledge software. Pressure recordings were begun and the proximal end of the catheter was advanced through the right atrium and ventricle into a pulmonary artery while monitoring the characteristic pulse pressures to identify the location (Owen et al., 1995b; Chapman and Wideman, 2001). The catheter was then slowly advanced until it was positioned in the pulmonary artery. Prior to recording, the system was calibrated for accuracy using a mercury manometer. After surgical preparations were complete and a 5-min stabilization had elapsed, control data were recorded for 5 min. Mannitol (2.5%, 0.1 mL/min per kg per BW) was infused through the PE-50 tubing in the left basilica vein to hydrate the bird (Wideman and Gregg, 1988) and act as a volume control and carrier vehicle. Serotonin (0.1 mg in 20 mL 2.5% mannitol) was infused at 0.1 mL/min per kg per BW through the PE-50 tubing in the left basilica vein for 10 min, after which 2.5% (wt/vol) mannitol infusion was resumed for a further 5 min. After a satisfactory recording was obtained, the birds were euthanized with 10 mL i.v. of 0.1 M KCl.
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FIGURE 2. Physiograph recording from an individual unanaesthetized male broiler in Experiment 2, showing continuous values for pulmonary arterial pressure (PAP) during a 5-min control period, after a 5-min infusion of 2.5% mannitol as a volume control, after a 10-min infusion of serotonin (0.1 mg in 20 mL 2.5% mannitol), followed by a further 5-min infusion of 2.5% mannitol.
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FIGURE 1. Physiograph recording from an individual male broiler in Experiment 1, showing continuous values for mean systemic arterial pressure (MAP), pulmonary arterial pressure (PAP), and blood flow through the right pulmonary artery (Flow) during a 5-min control period, after a 5-min infusion of 2.5% mannitol as a volume control, after a 10-min infusion of serotonin (0.1 mg in 20 mL 2.5% mannitol), followed by a further 5-min infusion of 2.5% mannitol.
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pressure returned toward pre-infusion base-line values. The pulmonary hypertensive responses of unanesthetized broilers were similar to those of anesthetized broilers (Figures 2 and 6).
DISCUSSION
FIGURE 3. Pulmonary arterial pressure (PAP, upper panel), pulmonary vascular resistance (PVR, middle panel), and blood flow through the right pulmonary artery (Flow, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-d) designate differences between means over time (P ≤ 0.05).
tended to decline as the 10-min infusion of 5HT progressed, but pulmonary arterial pressure remained elevated above baseline values throughout the interval of 5HT infusion. Blood flow through the pulmonary artery was depressed throughout the period of 5HT infusion (Figure 3). Contemporaneous reductions in cardiac output were associated with reductions in stroke volume but not with a reduction of heart rate (Figure 4). Mean systemic arterial pressure dropped below pre-infusion baseline levels during the pulmonary hypertensive responses to 5HT (Figure 5). Mean systemic arterial pressure and cardiac output responses also tended to be attenuated over the 10-min course of 5HT infusion but remained lower than the pre- or postinfusion baseline values. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial
FIGURE 4. Cardiac output (CO, upper panel), stroke volume (SV, middle panel), and heart rate (HR, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P ≤ 0.05); ns = not significant (P > 0.05).
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Any factor that reduces the pulmonary vascular capacity or increases the pulmonary vascular resistance theoretically can increase the incidence of PHS (Wideman and Bottje, 1993; Wideman, 2000). Serotonin infusion triggered rapid increases in pulmonary arterial pressure to approximately 50% above pre-infusion baseline values, accompanied by decreases in mean systemic arterial pressure and cardiac output, whereas carrier vehicle infusion alone had no influence on any of the hemodynamic variables. Pulmonary hypertensive responses were associated with increased pulmonary vascular resistance (pulmo-
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FIGURE 6. Pulmonary arterial pressure (PAP) for unanaesthetized male broilers (mean ± SEM, n = 9) in Experiment 2, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5-min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and at 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P ≤ 0.05).
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FIGURE 5. Mean systemic arterial pressure (MAP, upper panel), and total peripheral resistance (TPR, lower panel) for male broilers (mean ± SEM, n = 10) in Experiment 1, at the start of data collection (Start), at the start of the volume control infusion (C), at 5 min from the start of the volume control infusion (C5), within 30 s after serotonin infusion (SI), at 5 min intervals during serotonin infusion (SI5, SI10), within 30 s of ending serotonin infusion (EI), and at 5 min after ending serotonin infusion having resumed infusion of mannitol (EI5). Different letters (a-c) designate differences between means over time (P > 0.05); ns = not significant (P > 0.05).
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ACKNOWLEDGMENTS This research was supported by a US-Israel Binational Agricultural Research and Development grant (BARD US-2736-96) and by a grant from Hubbard ISA, Walpole, NH 03608.
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nary vasoconstriction). The peak pulmonary arterial pressure attainable was inadequate to propel the normal cardiac output through the elevated pulmonary vascular resistance. Consequently, the impeded venous return to the left ventricle caused dependant reductions in stroke volume, cardiac output, and mean systemic arterial pressure. Reductions in cardiac output were not associated with reductions in heart rate. The systemic hypotension that coincides with increased pulmonary vascular resistance reflects the inability of the relatively weak right ventricle to sustain a normal cardiac output. The peak pulmonary arterial pressure response occurred within approximately 70 s after the start of 5HT infusion, tended to decline over the course of a 10-min infusion period, but remained elevated above baseline values. The pulmonary hypertensive response to 5HT might have been partially attenuated by flow-dependent vasodilation mediated by the pulmonary vascular endothelium. Utilization of plasma L-arginine as a substrate for the production of the vasodilator nitric oxide might have relaxed the pre-existing tone of the pulmonary vessels, thereby allowing increased flow through the pulmonary vasculature at a lower pressure. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial pressure returned toward pre-infusion base-line values. The present study provides direct evidence that 5HT can trigger pulmonary vasoconstriction and pulmonary hypertension in broilers. The significance of this observation relates to the proposal that pulmonary hypertension may be triggered in susceptible broilers by poor air quality and respiratory disease. Pulmonary endothelial-cell damage has been associated with platelet activation in humans, resulting in increased release of 5HT (Dekker, 1995). Furthermore, elevated plasma concentrations of 5HT have been implicated in the pulmonary hypertension associated with acute respiratory disease syndrome in humans (Heffner and Repine, 1997). Lungs of broilers developing ascites contain accumulations of mast cells, macrophages, and inflammatory foci (Maxwell et al., 1986, 1989; Owen et al., 1995a). In addition to antigens entering the lungs through inspired air, venous blood returning to the right ventricle of the heart also creates a continuous pulmonary exposure to blood-borne particles. In one study, bolus intravenous injections of bacterial endotoxin were shown to trigger pulmonary vasoconstriction and pulmonary hypertension in broiler chickens (Wideman et. al., 2001). This process can be profoundly damaging to pulmonary hemodynamics and gas exchange and may contribute to the pathogenesis of PHS.
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