Evaluation of the Serotonin Receptor Blocker Methiothepin in Broilers ...

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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Evaluation of the Serotonin Receptor Blocker Methiothepin in Broilers Injected Intravenously with Lipopolysaccharide and Microparticles1 M. E. Chapman2 and R. F. Wideman Jr. Department of Poultry Science, University of Arkansas, Fayetteville 72701 ABSTRACT There has been considerable interest in the role of serotonin (5-hydroxytryptamine, 5-HT) in the pathogenesis of pulmonary hypertension due to episodes of primary pulmonary hypertension in humans linked to serotoninergic appetite-suppressant drugs. In this study, we investigated the effect of 5-HT on the development of pulmonary hypertension induced by injecting bacterial lipopolysaccharide (LPS; endotoxin) and cellulose microparticles intravenously, using the nonselective 5-HT¹⁄₂ receptor, antagonist methiothepin. In Experiment 1, broilers selected for ascites susceptibility or resistance under conditions of hypobaric hypoxia were treated with methiothepin or saline, followed by injection of LPS, while recording pulmonary arterial pressure (PAP). In Experiment 2 ascites-susceptible broilers were treated with methiothepin or saline, followed by injection of cellulose microparticles, while recording PAP. In Experiment 3, an i.v. microparticle injection dose shown to cause 50% mortality was injected into ascites-susceptible and ascitesresistant broilers after methiothepin or saline treatment. Injecting methiothepin reduced PAP below baseline val-

ues in ascites-susceptible and ascites-resistant broilers, suggesting a role for 5-HT in maintaining the basal tone of the pulmonary vasculature in broilers. Injecting microparticles into the wing vein had no affect on the PAP in the broilers treated with methiothepin, suggesting that 5HT is an important mediator in the pulmonary hypertensive response of broilers to microparticles. Furthermore, injecting an 50% lethal dose of microparticles into ascitessusceptible and ascites-resistant broilers pretreated with methiothepin resulted in reduced mortality. Serotonin appears to play a less prominent role in the pulmonary hypertensive response of broilers to intravenously injected LPS, indicating that other mediators within the innate response to inflammatory stimuli may also be involved. These results are consistent with our hypothesis that pulmonary hypertension syndrome ensues when vasoconstrictors, such as 5-HT, overwhelm the dilatory effects of vasodilators, such as NO, thereby effectively reducing the pulmonary vascular capacity of pulmonary hypertension syndrome-susceptible broilers.

Key words: broiler, serotonin, lipopolysaccharide, microparticle, methiothepin 2006 Poultry Science 85:2222–2230

INTRODUCTION Fast-growing broilers are susceptible to pulmonary hypertension, leading to pulmonary hypertension syndrome (PHS, ascites) when their right ventricle must progressively elevate the pulmonary arterial pressure (PAP) to propel the requisite cardiac output through lungs having a marginally inadequate pulmonary vascular capacity (Peacock et al., 1989; Julian, 1993; Wideman and Bottje, 1993; Wideman, 2000, Wideman et al., 2001). Any factor

2006 Poultry Science Association Inc. Received March 10, 2006. Accepted May 9, 2006 1 US patent 6,720,473 protects the exclusive rights of the University of Arkansas to all uses of the i.v. microparticle injection technology within the context of evaluating or affecting pulmonary vascular capacity, pulmonary vascular resistance, pulmonary hypertension, cardiopulmonary hemodynamics, and susceptibility to pulmonary hypertension and pulmonary hypertension syndrome (ascites) in domesticated animal species. 2 Corresponding author: [email protected]

that increases the cardiac output, reduces the pulmonary vascular capacity, or triggers pulmonary vasoconstriction can contribute to the pathogenesis of PHS in broilers (Wideman, 2000). There has been considerable interest in the role of serotonin (5-hydroxytryptamine, 5-HT) in the pathogenesis of pulmonary hypertension due to episodes of primary pulmonary hypertension in humans linked to serotoninergic appetite-suppressant drugs (Abenhaim et al., 1996). Increased plasma 5-HT levels have also been recorded in pulmonary hypertensive human patients (Herve et al., 1995). Serotonin is a potent pulmonary vasoconstrictor 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). Serotonin has been implicated in the mechanisms responsible for pulmonary hypertension in several human, animal, and broiler studies (Seiler et al., 1974; Douglas et al., 1981; Brenot et al., 1993; Abenhaim et al., 1996; Chapman and Wideman, 2002).

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When platelets and thrombocytes aggregate, they release several physiologically active substances, including 5-HT, which causes proliferation of pulmonary vascular smooth muscle and stimulates 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). The pulmonary vasoconstriction triggered by 5-HT is believed to be mediated through 5-HT1B/1D and 5-HT2A receptors expressed by pulmonary smooth muscle cells (Choi and Maroteaux, 1996; MacLean et al., 1996). A previous study (Chapman and Wideman, 2006) provided direct evidence that methiothepin can inhibit the hypertensive response to 5-HT in broilers and that this was associated with increased pulmonary vascular resistance (pulmonary vasoconstriction) rather than an increase in cardiac output. Methiothepin is a nonselective 5-HT1 and 5-HT2 as well as a 5-HT5-7 receptor antagonist with varying degrees of selectivity. However, methiothepin displays high affinities for 5-HT1A and 5-HT1B receptor subtypes in rats (Engel et al., 1986). The selective 5-HT2A antagonist ketanserin has been used successfully in humans to lower blood pressure in hypertensive patients (Vanhoutte et al., 1988). However, ketanserin fails to attenuate the pulmonary hypertensive responses to infused 5-HT, suggesting that the 5-HT2A receptor is not important in modulating the hypertensive response to 5-HT in broilers (Chapman and Wideman, 2006). Therefore, in the present study, methiothepin was used as a tool for evaluating the role of 5-HT in the onset of pulmonary hypertension triggered by inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) and cellulose microparticles in broilers. We investigated the effect of methiothepin on the development of pulmonary hypertension induced by injecting bacterial LPS and cellulose microparticles intravenously in broilers. In Experiment 1, broilers selected for ascites susceptibility and resistance under conditions of hypobaric hypoxia were treated with methiothepin or saline, followed by injection of LPS, while recording PAP. Lipopolysaccharide causes pulmonary vasoconstriction and pulmonary hypertension in broilers (Wideman et al., 2001), but the specific mediator of vasoconstriction has not been determined. The objective of Experiment 1 was to determine if using methiothepin to block the vasoconstrictor response to 5-HT would attenuate the pulmonary hypertensive response to LPS. In Experiment 2, ascites-susceptible broilers were treated with methiothepin or saline, followed by injection of cellulose microparticles, while recording PAP at a dose designed to cause pulmonary hypertension without causing mortality. Microparticles cause pulmonary vasoconstriction in broilers (Wideman and Erf, 2002), but the specific mediator of vasoconstriction has not been determined. The objective of Experiment 2 was to determine if using methiothepin to block the vasoconstrictor response to 5-HT would attenuate the pulmonary hypertensive response to microparticles. To further evaluate the responses to microparticles after treatment with saline and methiothepin, mortality caused by an i.v. microparticle injection dose previously demonstrated to cause 50% mortality (LD50) was recorded in ascites-susceptible and ascites-re-

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sistant broilers in Experiment 3. Intravenous microparticle injections can be used to eliminate broilers having the most restrictive pulmonary vasculature. The venous blood carries the microparticles to the lungs where they occlude pulmonary arterioles in proportion to the numbers of microparticles injected. Broilers with the most limited pulmonary vascular capacity succumb to respiratory insufficiency within 24 h. Theoretically, broilers with the most robust pulmonary vascular capacity survive primarily because sufficient vascular channels remain unoccluded to convey the cardiac output without requiring an excessive increase in PAP (Wideman et al., 2002, 2006). In addition to physically occluding pulmonary arterioles, entrapped microparticles can stimulate local tissues and leukocytes to release vasoactive substances capable of altering pulmonary vascular resistance by dilating or constricting the nearby vasculature (Wang et al., 2003; Wideman et al., 2004).

MATERIALS AND METHODS Strategies for selecting ascites-susceptible and ascitesresistant broiler lines under conditions of hypobaric hypoxia have been described previously (Anthony et al., 2001; Balog et al., 2003). After the eighth generation of selection, progeny of the susceptible and resistant lines exhibited ascites mortalities of 98.6% and 26.0%, respectively, when grown under hypobaric conditions (Pavlidis, 2003). Male progeny from the 10th generation of these lines were wingbanded and transported on the day of hatch (d 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 d 1 to 5, 29°C from d 6 to 10, and 27°C from d 11 to 17. Thereafter, the broilers were maintained at 23.8°C until the experiment was terminated. The photoperiod was 24 h of light from d 1 to 5, and 23L:1D thereafter. Water was provided ad libitum via nipple-type waterers. A corn–soybean meal starter ration (22.7% CP, 3,059 kcal ME/kg, 1.5% Arg, and 1.43% Lys) was provided ad libitum and was formulated to meet minimum NRC (1994) standards for all ingredients. The diet was provided as crumbles during wk 1 and 2 and as pellets thereafter. All surgeries were completed from d 28 to 42.

Experiment 1 Male broilers from the resistant (n = 24; 2,033 ± 70.5 g of BW, mean ± SEM) and susceptible lines (n = 20; 2,226 ± 72.4 g of BW, mean ± SEM) were anesthetized to a light surgical plane with i.m. injections of allobarbitol [5,5diallylbarbituric acid (Sigma Chemical Co., St Louis, MO) 3.0 mL of 5,5-diallylbarbituric acid, 25 mg/mL) and ketamine HCl (Bedford Laboratories, Bedford, OH; 1.0 mL, 100 mg/mL). The birds were placed on a heated surgical board (30°C) and restrained in dorsal recumbency. The left wing was extended, and feathers were removed from the ventral surface as needed to uncover the skin over the basilica vein. After intracutaneous injections of 2% lido-

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caine HCl were administered as a local anesthetic, an incision was made to expose the vein, which then was cannulated with a silastic catheter (0.012 in inside diameter., 0.037 in outside diameter.) filled with a 0.9% NaCl solution containing 200 IU heparin/mL. The catheter was attached to a blood pressure transducer interfaced through a Transbridge preamplifier (World Precision Instruments, Sarasota, FL) to a Biopac MP 100 data acquisition system using AcqKnowledge software (Biopac Systems Inc., Goleta, CA). The catheter was advanced through the right atrium and ventricle into a pulmonary artery while monitoring the characteristic pulse pressures to identify the location (Wideman et al., 1996; Chapman and Wideman, 2001). All PAP readings were made with the transducer at the level of the thoracic inlet. Before recording, the system was calibrated in millimeters of Hg (mm Hg) using a Hg manometer. The left basilica vein was also cannulated with PE-50 polyethylene tubing filled with heparinized saline for i.v. injections. After surgical preparations were complete and a stabilization period of 10 min had elapsed, control data were recorded for 10 min. Ascites-susceptible and ascitesresisistant broilers were then injected with 3 mg/kg of BW methiothepin mesylate salt (Sigma Chemical Co.) in 0.9% saline (n = 22) or 0.9% saline alone (n = 22) and data were recorded for a further 10 min. This dose of methiothepin previously was demonstrated to fully inhibit the pulmonary hypertensive response to i.v. 5-HT infusion in broilers (Chapman and Wideman, 2006). Next, the broilers were injected with 1 mg of Salmonella typhimurium LPS (dissolved at 2 mg/mL in 0.9% NaCl; Sigma Chemical Co.) through the PE-50 tubing in the left basilica vein. Data were recorded for a further 40 min, after which the birds were euthanized intravenously with 10 mL of 0.1 M KCl.

Experiment 2 Ascites-susceptible male broilers (n = 15; 2,663 ± 118.5 g of BW, mean ± SEM) were anesthetized and cannulated for measurement of PAP and i.v. infusions, as described above. After surgical preparations were complete and a stabilization period of 10 min had elapsed, control data were recorded for 10 min. Broilers were then injected with 3 mg of methiothepin (n = 8) or 0.9% saline (n = 7)/kg of BW, and data were recorded for a further 10 min. Treatment was followed by injection of 0.35 mL of microgranular CM-32 ion exchange cellulose microparticles (Fisher Scientific, St. Louis, MO) suspended at 0.02 g/mL in heparinized saline through the PE-50 tubing in the left basilica vein. Previous studies have demonstrated that approximately 0.3 to 0.35 mL of the 0.02 g/mL microparticle suspension is required to significantly elevate the PAP while triggering 0.05) initial PAP values in the ascitessusceptible line, when compared with the resistant line before methiothepin or saline injection (St to S10), the per-

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Figure 1. Pulmonary arterial pressure (PAP, panel A) and percentage of change from the initial PAP (PAP % change, panel B) for male broilers (n = 20; 2,226.3 ± 72.4 g of BW, mean ± SEM) in Experiment 1 at the start of data collection and at intervals of 5 min thereafter (St, S5, S10) within 30 s after methiothepin or saline injection and at intervals of 5 min thereafter (M, M5, M10) within 30 s after lipopolysaccharide (LPS) injection and at intervals of 5 min thereafter (L to L40). a–cLetters represent differences (P ≤ 0.05) between groups within individual sample intervals. Asterisks (*) represent differences (P ≤ 0.05) within a group across sample intervals.

centage of change from the initial PAP is shown in Figure 1, panel B, for comparisons between the groups. Injecting methiothepin caused the PAP to drop by approximately 25% in relation to the initial PAP in both the ascites-susceptible and ascites-resistant lines, whereas saline injection

had no effect on the percentage of change in PAP in relation to the initial PAP in either line. The PAP in both susceptible and resistant lines injected with methiothepin remained depressed below the initial PAP and saline-injected lines at all sample intervals up to 5 min post-LPS injection.

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Table 1. Values for ∆ change in pulmonary arterial pressure (PAP) for male broilers selected for ascites resistance1 and susceptibility2 in Experiment 1 during control sample intervals (control) after an i.v. injection of saline or methiothepin (M to M10). Sample interval3 Variable PAP PAP PAP PAP

Group Resistant saline Susceptible saline Resistant methiothepin Susceptible methiothepin

Control 0 0 0 0

M −0.1 −0.3a −2.2*b −5.2*c a

M5

M10

−0.4 −0.2a −1.7b −4.4*c

−0.3 −0.2a −2.1b −4.5*b

ab

a

L a

0 0.5a −2.0b −4.2*b

L5

L10 b

0.4 3.5a −2.1bc −5.4*c

L15

b

b

0.9 8.8*a −1.8*b −2.4b

2.5 5.3* 0.6b −1.6b

L20 7.8* 8.3* 5.1 3.6*

L25 8.5* 10.5* 7.6* 6.4*

L30

L35

L40

5.3* 8.9* 5.3* 5.7*

ab

0.6b 6.2*a 0.2b 2.3ab

3.2 7.4*a 2.2b 4.4*ab

Differences (P ≤ 0.05) within a sample interval. Values are means for 10 broilers per group. 2 Values are means for 12 broilers per group. 3 Sample intervals were approximately 5 min apart. *The means within a row differed (P ≤ 0.05) from the control value by repeated measures ANOVA. a–c 1

Injecting 1 mg of LPS into the wing vein caused the PAP to rise and reach a peak within 25 min post-LPS injection in all groups, regardless of line (susceptible, resistant) and treatment (saline, methiothepin), and whereas the PAP tended to be lower in the susceptible and resistant broilers treated with methiothepin, at no sample interval during the main hypertensive response to LPS (L20 to L35) did the percentage of change in PAP differ between the groups (P > 0.05).

Experiment 2 As shown in Figure 2, panel A, the initial PAP averaged approximately 23 mm Hg for ascites-susceptible broilers in the methiothepin-treated group and approximately 18 mm Hg for susceptible broilers in the saline-injected group. The delta changes in PAP from baseline values are shown in Table 2. Due to the higher (P > 0.05) initial PAP values in the methiothepin-treated broilers before methiothepin or saline injection (St, S5), the percentage of change from the average initial PAP is shown for each group in Figure 2, panel B. Injecting methiothepin caused the PAP to drop in relation to the initial PAP by approximately 25%, whereas saline injection had no effect on the percentage of change in PAP in relation to the initial PAP. Injecting cellulose microparticles into the wing vein caused the PAP to rise by approximately 80% within 5 min in the broilers treated with saline as a volume control, reaching a peak PAP of approximately 90% above initial PAP within 10 min postmicroparticle injection. The pulmonary hypertensive response remained elevated by approximately 70% above baseline values for the remainder of the experiment. Injecting microparticles into the wing vein had no effect on the PAP in the broilers treated with methiothepin.

Experiment 3 Mortality data are shown in Figure 3. Injecting cellulose microparticles caused 78% mortality within 24 h in the ascites-susceptible broilers, whereas injecting methiothepin 10 min before microparticles in susceptible broilers reduced the mortality to 20%. Injecting methiothepin 10 min before microparticles eliminated the mortality (0%) in the ascites-resistant broilers compared with 12% mortality in resistant broilers injected with microparticles alone.

DISCUSSION Injecting the nonselective 5-HT1/2 receptor antagonist, methiothepin, reduced PAP below baseline values by approximately 25% in the ascites-susceptible and ascites-resistant broilers in Experiments 1 and 2, suggesting a role for 5-HT in maintaining the basal tone of the pulmonary arterial wall in broilers. Injecting 1 mg of LPS into the wing vein in Experiment 1 caused PAP to rise within 20 min in the ascites-susceptible and ascites-resistant lines injected with saline as a volume control and within 25 min in the ascites-susceptible and ascites-resistant lines treated with methiothepin. The peak PAP response to LPS was attained within 25 min postinjection in all groups, regardless of line and treatment, suggesting that 5-HT may not play a prominent role in the pulmonary hypertensive response to LPS in broilers. Thromboxane A2 (TxA2), whether administered intravenously as the potent TxA2 mimetic U44069 or produced by circulating thrombocytes in response to bolus acid injections, has been shown to increase pulmonary vascular resistance and PAP in broilers (Wideman et al., 1998, 1999, 2001), and several studies have indicated that the pulmonary hypertension in endotoxemia is due to TxA2. Levels of thromboxane B2 (a stable metabolite of TxA2) in plasma and lung lymph increased acutely after LPS administration in several mammalian species (Casey et al., 1982; Watkins et al., 1982; Ball et al., 1983; Snapper et al., 1983; Winn et al., 1983; Kubo and Kobayashi, 1985; Ogletree et al., 1986;). Furthermore, pretreatment with a cyclooxygenase inhibitor (Ogletree and Brigham, 1982; Snapper et al., 1983) blocks the rise in thromboxane B2 levels and the acute pulmonary hypertension associated with endotoxemia in mammals. Definitive studies have yet to be conducted to evaluate the potential role of TxA2 in the pulmonary hypertensive response to LPS in broilers. An early hypertensive peak that rarely develops in broilers was recorded 10 min post-LPS injection in the ascitessusceptible broilers treated with saline. Attenuation of this early peak by treatment with methiothepin suggests the involvement of 5-HT. Broilers vary little in their pulmonary vascular responsiveness to 5-HT (Chapman and Wideman, 2002); however, no early (10 min post-LPS) hypertensive response was evident in the ascites-resistant broilers. This

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Figure 2. Pulmonary arterial pressure (PAP, panel A) and percentage of change from the initial PAP (PAP % change, panel B) for male broilers (n = 15; 2,662.9 ± 118.5 g of BW, mean ± SEM) in Experiment 2 at the start of data collection and at intervals of 5 min thereafter (St, S5, S10) within 30 s after methiothepin or saline injection and at intervals of 5 min thereafter (M, M5, M10) within 30 s after microparticle injection and at intervals of 5 min thereafter (L to L40). a,bLetters represent differences (P ≤ 0.05) between groups within individual sample intervals. Asterisks (*) represent differences (P ≤ 0.05) within a group across sample intervals.

indicates that innate factors, such as 5-HT release by thrombocytes, 5-HT receptor density, or modulation by vasodilators, such as NO, may be responsible for the individual responsiveness of broilers to LPS. This early hypertensive response to LPS has been observed commonly in broilers treated with the NO synthase (NOS) inhibitor N ω-nitro-

L-arginine methyl ester, suggesting a role for NO in modulating the early hypertensive response to LPS in broilers (Wideman et al., 2001; Wang et al., 2002a). It seems likely, therefore, that the hypertensive responses to LPS in broilers are the result of the interaction of vasodilators (NO) and vasoconstrictors (5-HT and, possibly, TxA2). Nitric oxide

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Table 2. Values for ∆ change in pulmonary arterial pressure (PAP) for mail broilers selected for ascites susceptibility in Experiment 2 during control sample intervals (Control) after an i.v. injection of saline1 or methothepin2 (M to M10) and after an i.v. injection of microparticles (L to L40). Sample interval3 Variable PAP PAP

Group Susceptible saline Susceptible methiothepin

Control 0 0

M a

0.4 −5.2*b

M5

M10

0.1 −2.5

−0.1 −2.3

L

L5 a

4.9* −0.2b

L10 a

13.4* 1.6b

L15 a

14.6* 3.0b

L20 a

14.1* 3.6b

L25 a

13.1* 2.8b

L30 a

12.5* 2.2b

L35 a

12.4* 1.8b

L40 a

12.5* 1.4b

11.4*a 0.8b

Differences (P ≤ 0.05) within a sample interval. Values are means for 7 broilers. 2 Values are means for 8 broilers. 3 Sample intervals were approximately 5 min apart. *The means within a row differed (P ≤ 0.05) from the control value by repeated measures ANOVA. a,b 1

also reduces or inhibits the LPS-stimulated release of TxA2 and 5-HT, thereby minimizing vasoconstriction attributable to thrombocyte activation (Longworth et al., 1994; Frank et al., 1996; Gaston and Stamler, 1997; Teder and Nobel, 2000; Davis and Matalon, 2001; Gryglewski et al., 2001; Miyata et al., 2001; Lauer et al., 2002). Lipopolysaccharide induces multiple cell types to express the gene for NOS (Chang et al., 1996; Dil and Qureshi, 2002a,b; Janeway and Medzhitov, 2002; Qureshi, 2003). Endothelial NOS (eNOS) can be induced in endothelial cells to produce NO transiently (Szabo´, 1995) and likely accounts for the attenuation of the pulmonary hypertensive responses of broilers to LPS and microparticles seen toward the end of Experiments 1 and 2 (Bowen et al., 2006; Wideman et al., 2006). In Experiment 2, i.v. cellulose microparticle injection triggered a substantial increase in PAP in broilers treated with saline as a volume control. Broilers pretreated with methiothepin failed to exhibit a significant pulmonary hypertensive response to microparticle injection, indicating

Figure 3. Percentage of mortality for ascites-susceptible (n = 57; 2,659.6 ± 36.7 g of BW, mean ± SEM) and ascites-resistant (n = 78; 2,680.0 ± 35.6 g of BW, mean ± SEM) male broilers in Experiment 3 after injection of an 50% lethal dose of microparticles with and without prior treatment with methiothepin. ab,xLetters represent differences (P ≤ 0.05) between groups. Numbers within bars represent the number of birds that died over the total number of birds in that group.

that 5-HT is an important mediator in the pulmonary hypertensive response of broilers to microparticles. Furthermore, injecting microparticles at a dose shown to cause LD50 in unselected broilers resulted in 78% mortality within 24 h in the ascites-susceptible broilers in Experiment 3, whereas injecting methiothepin 10 min previously to microparticles in susceptible broilers reduced mortality to 20%. Injecting an LD50 dose of microparticles for unselected broilers 10 min after methiothepin resulted in 0% mortality within 24 h in the ascites-resistant broilers as compared with 12% mortality in resistant broilers injected with microparticles alone. It has been shown that within minutes after being injected, the entrapped microparticles are surrounded by focal aggregates of thrombocytes and monocytes, macrophages, or both (Wideman et al., 2002; Wang et al., 2003), which potentially can trigger these leukocytes and the adjacent vascular endothelium to synthesize and release potent vasoactive compounds within close proximity to the vascular smooth muscle (Wideman, 2000; Wideman et al., 2004). The tendency for the PAP to increase slightly in broilers treated with methiothepin after microparticle injection may reflect physical occlusion of precapillary arterioles or may be due to the influence of other inflammatory mediators, such as TxA2. The tendency for the pulmonary hypertensive response to microparticles to become attenuated toward the end of the experiment, despite intravenously injected cellulose microparticles being shown to persist in the pulmonary microvasculature for more than 10 d (Wideman et al., 2002; Wang et al., 2003), may be attributable to an increase in NO synthesis. Increased rates of blood flow through unoccluded vascular channels would be expected to stimulate constitutive eNOS within the vascular endothelium to generate NO as a modulator of flow-dependent pulmonary vasodilation (Wideman et al., 1995, 1996, 1998). Indeed, recent studies have shown that inhibition of NOS by N ω-nitro-L-arginine methyl ester exposed a more dramatic pulmonary vasoconstriction and hypertension in response to a standard dose of intravenously injected microparticles in broilers (Wideman et al., 2005b). In summary, this study provides direct evidence that 5HT plays an important role in maintaining the basal tone of the pulmonary vasculature in broilers and in the pulmonary hypertensive response of broilers to intravenously injected microparticles. Microparticle injections are used

EVALUATION OF THE SEROTONIN RECEPTOR BLOCKER METHIOTHEPIN

to successfully eliminate PHS-susceptible broilers from key genetic lines (Wideman et al., 2002); consequently, the likelihood exists that microparticle selection serves to eliminate individuals having an excessive production, release, or responsiveness to 5-HT. Serotonin appears to play a less prominent role in the pulmonary hypertensive response of broilers to intravenously injected LPS, indicating that other mediators within the innate response to inflammatory stimuli, possibly TxA2, may also be involved. This is consistent with our hypothesis that PHS ensues when vasoconstrictors overwhelm the dilatory effects of vasodilators such as NO. Further study will be required to ascertain whether the complex interactions between these mediators of the innate inflammatory response are a result of their altered synthesis, release, or the number of receptors for these mediators available.

ACKNOWLEDGMENTS This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, USDA/CSREES/NRI grant no. 2003-35204-13392, and by an animal health grant from the University of Arkansas Agricultural Experiment Station.

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