Methysergide augments the acute, but not the sustained, hypoxic ventilatory response in goats

Respiration Physiology 118 (1999) 25 – 37 www.elsevier.com/locate/resphysiol

Methysergide augments the acute, but not the sustained, hypoxic ventilatory response in goats Jay K. Herman *, Ken D. O’Halloran, Gordon S. Mitchell, Gerald E. Bisgard Department of Comparati6e Biosciences, School of Veterinary Medicine, Uni6ersity of Wisconsin-Madison, 2015 Linden Dri6e West, Madison, WI 53706, USA Accepted 12 July 1999

Abstract Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation that occurs during sustained hypoxia. As serotonin (5-HT) has been reported to be an important modulator of respiratory output, 5-HT may also play a role in VAH. Methysergide (a broad-spectrum 5-HT antagonist), was given to awake goats (1 mg kg − 1 i.v.) 30 min prior to being exposed to 4 h of isocapnic hypoxia. Although methysergide slightly decreased arterial pH, presumably due to a non-significant increase in arterial PCO2, it did not alter normoxic ventilation. Following methysergide, the expired minute ventilation (V: E) was significantly elevated above the control (saline) response after 30 min of hypoxia, but methysergide did not otherwise alter VAH. We repeated the study in the same goats using ketanserin, a specific 5-HT2A/2C receptor antagonist (1.2 mg kg − 1 i.v.). Ketanserin had no effect on the acute hypoxic ventilatory response, or on VAH. We conclude that while 5-HT modulates the acute hypoxic ventilatory response in goats, it does not appear to act through the 5-HT2A/2C receptor subtypes. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Control of breathing, ventilatory acclimatization to hypoxia; Hypoxia, ventilatory acclimatization, serotonin; Mammals, goat; Mediators, serotonin; Pharmacological agents, methysergide

1. Introduction Prolonged exposure to low inspired oxygen levels results in a time-dependent increase in ventilation that is termed ventilatory acclimatization to hypoxia (VAH). This increased ventilatory output * Corresponding author. Tel.: +1-608-2622962; fax: +1608-2633926. E-mail address: [email protected] (J.K. Herman)

occurs despite a constant level of isocapnic hypoxia (Bisgard and Neubauer, 1995). Earlier reports indicate that VAH requires intact carotid body chemoreceptors (Smith et al., 1986), that carotid body hypoxia is sufficient to elicit VAH (Busch et al., 1985; Dwinell et al., 1997b), and that carotid chemo-afferent neurons progressively increase their firing frequency during VAH (Nielsen et al., 1988). Thus, VAH appears to result primarily from incremental carotid body chemoreceptor output, at least during 4 h of

0034-5687/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 4 - 5 6 8 7 ( 9 9 ) 0 0 0 7 0 - 5

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hypoxia in goats. However, this conclusion does not rule out the possibility that some changes in respiratory motor output during VAH may result from changes in CNS integration of carotid chemo-afferent inputs. Serotonin (5-HT) has been reported in the carotid bodies of many mammalian species (Gonzalez et al., 1995), including goats (Engwall et al., 1989). While 5-HT does not appear to play a significant role in the acute carotid body response to hypoxia (Kirby and McQueen, 1984), the role of carotid body 5-HT during VAH is unknown. There is compelling evidence implicating 5-HT as an important modulator of other respiratory behaviors besides VAH (McCrimmon et al., 1995). For example, certain forms of plasticity in respiratory responses have been reported to be dependent on 5-HT receptor activation (Millhorn et al., 1980; Bach et al., 1993; Bach and Mitchell, 1996); including long-term facilitation of respiratory motor output following episodic hypoxia, a mechanism suggested to play a role in VAH (Millhorn et al., 1980). The role of 5-HT in VAH was not supported by the findings of Olson (1987), who reported no change in the time course of VAH in rats treated with p-chlorophenylalanine (PCPA) to systemically deplete 5-HT levels. However, PCPA induced 5-HT depletion may not be as effective as 5-HT receptor antagonists in blocking the effects of 5-HT on respiratory motor output (Millhorn et al., 1980). While it is apparent that VAH involves a progressive increase in carotid body discharge, which results in, or is coincidental with an increase in respiratory motor output, the mechanisms that underlie these changes are not well known. To test the hypothesis that 5-HT is necessary for the facilitation of ventilation associated with VAH, we used an established model of VAH (exposing awake goats to 4 h of sustained isocapnic hypoxia). Initially, methysergide was used to induce systemic 5-HT receptor blockade. Although methysergide is non-selective, it has been reported to be a potent antagonist at the 5-HT2A/2C receptor subtypes (Hoyer et al., 1994). Furthermore, both methysergide and ketanserin have been reported to antagonize the 5-HT induced excitation of spinal motor neurons (Jackson and White,

1990). Therefore, this study was repeated using the more selective 5-HT2A/2C receptor antagonist, ketanserin, to determine if the response elicited by methysergide was mediated via the 5-HT2A/2C receptor subtypes.

2. Methods

2.1. Animal preparation Six adult female goats [mean body weight 549 7 kg (9 SD)] were used in this study. After initial anesthetic induction (15 mg kg − 1 sodium thiopental i.v.), the goats were maintained under general anesthesia (halothane, nitrous oxide and oxygen) while one common carotid artery was translocated to a subcutaneous position to facilitate insertion of an arterial catheter. Following the surgery, during a minimum 2-week-recovery period, each goat was trained to stand quietly in a stanchion while wearing a facemask. One day prior to the study, an arterial catheter was inserted into the translocated carotid artery for anaerobic blood sampling and blood pressure measurement. A venous catheter was also placed in an external jugular vein for i.v. drug administration.

2.2. Measurements Ventilatory data were collected with a tightly fitting facemask attached to a low resistance unidirectional breathing valve (Hans Rudolph, no. 2700). Inspired gases were delivered to the goat via (3 cm, i.d.) flexible tubing. Expired gases were collected in a spirometer (120 l) from which expired minute ventilation (V: E) could be measured throughout the experimental protocol. Inspired airflow was measured using a pneumotachograph (T-2, Fleisch) which was electronically integrated to give inspired tidal volume. An O2 analyzer (Applied Electrochemistry, S-3A) was used to monitor the concentration of O2 in the inspired gases. Inspired and expired CO2 levels were measured from a port in the facemask using a CO2 analyzer (PM-20, Anarad).

J.K. Herman et al. / Respiration Physiology 118 (1999) 25–37

Initially, a six-channel polygraph was used to record end-tidal CO2, systemic arterial blood pressure, integrated inspired volume and expired minute volume. After the first four animals, the remaining experiments were recorded using the Windaq data acquisition system (DATAQ Instruments, Akron OH). Each variable was collected at 50 – 100 Hz channel – 1 and data were collected continuously over the entire experimental protocol. Only the ventilatory and cardiovascular variables measured at times corresponding to blood gas measurements are presented in this paper. Arterial blood pressure and blood gas samples were obtained via a catheter in the carotid loop. Arterial blood samples were analyzed for arterial pH, PCO2 and PO2 (pHa, PaCO2, PaO2, respectively) by blood gas analyzer (ABL 500, Radiometer). A rectal thermistor probe was used for measurement of body temperature to correct blood gas measurements for temperature.

2.3. Protocol The goats were allowed : 20 – 30 min to adapt to the breathing circuit. Once stable baseline variables were established, 30 min of control respiratory, cardiovascular and blood gas variables were collected. At the end of this period, the goats were given either methysergide maleate (1 mg kg − 1 i.v.) or vehicle (12 – 14 ml saline i.v.) and an additional 30 min of baseline variables were collected. Following this second control period, the inspired gas mixture was adjusted until a stable level of hypoxia was achieved (PaO2 of 40.590.5 torr for both groups). Inspired CO2 was added and continuously adjusted to maintain PaCO2 levels isocapnic to the post-injection baseline PaCO2. The goats were maintained at this isocapnic PaO2 level for 4 h. Arterial blood samples were taken at a minimum of every 15 min to ensure that there were no fluctuations in arterial blood gases. For each time point during the experimental protocol, 2 – 5 blood gas samples were measured and the ventilatory and cardiovascular variables corresponding to these samples were analyzed. At the end of this 4 h period, the goats were allowed to breathe room air without added inspired CO2 for : 30 –60 min. Following this period the inspired

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O2 was again lowered until the PaO2 reached 39.79 0.4 torr (both groups) to test the postVAH acute hypoxic ventilatory response. Inspired CO2 was added to maintain the PaCO2 isocapnic to the post-VAH normoxic PaCO2 level. The goats were allowed a minimum of 2 weeks recovery before the protocol was repeated with the opposite injection (methysergide maleate or saline). At least 1 month following the conclusion of the above protocol, the experiment was repeated in the same goats using the more specific 5-HT2 receptor antagonist, ketanserin tartrate (Hoyer et al., 1994). Ketanserin was administered in the molar equivalent concentration given for the methysergide maleate (1.2 mg kg − 1 i.v. in 12–14 ml saline). The protocol was identical to that for methysergide except for the duration of the hypoxic exposure. The goats were exposed to 3 h isocapnic hypoxia (PaO2 = 40.69 0.7 torr).

2.4. Drugs The methysergide maleate was kindly donated to Dr. Gordon Mitchell by Sandoz as per Bach and Mitchell (1996). Ketanserin tartrate was purchased from Research Biochemicals Inc. (RBI, Natick, MA). Both the methysergide and ketanserin concentrations were determined using the salt weight and then dissolved in 12 –14 ml saline before being slowly administered to the goats (: 2 min infusion, i.v.).

2.5. Statistical analysis Expired minute ventilation, f, VT, heart rate (fH), and blood pressure were collected immediately preceding blood gas measurements during the experimental protocol. The variables reported are the average of 2–5 blood gas measurements taken during the baseline and at specific time points during hypoxia. A two-way, repeated-measures ANOVA was used to test for significant differences among the different time points and between drug treatments. Post-hoc analysis using the Tukey HSD test was used where indicated by a significant ANOVA. All statistical tests were performed using Statistica (StatSoft, version 4.5). The level of significance was set at PB 0.05. All data are presented as means 91 SE.

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3. Results

3.1. Control Bolus injections of saline (12 – 14 ml i.v.) did not induce any change in the arterial blood gas variables (Table 1). Lowering the inspired O2 tension significantly lowered PaO2 to approximately 40 torr throughout the 4 h protocol. Arterial PCO2 was not changed significantly at any time during the 4 h acclimatization period. Arterial pH decreased slightly during the period of sustained hypoxia due to a slight decrease in HCO− 3 (mmol l − 1; Table 1). Saline injections did not alter normoxic ventilation (Fig. 1). Isocapnic hypoxia significantly increased f (Fig. 1A), VT (Fig. 1B) and V: E (Fig. 1C). All ventilatory variables increased in a timedependent manner during hypoxia and were significantly elevated above the acute hypoxic response at the second through fourth hour of isocapnic hypoxia (Fig. 1). Upon return to room air, all ventilatory variables returned to the prehypoxia control levels, although there was a significant decrease in mean PaCO2 of 2.8 9 0.7 torr (PB 0.05). There were no differences in pHa, PaO2 or HCO− between the pre- and post-hypoxia 3 normoxic (or control) time points (Table 1).

3.2. Methysergide Blood gases and pHa in the methysergidetreated goats are summarized in Table 1. Methysergide had no significant effects on PaO2 or HCO− 3 . Thirty minutes following methysergide administration, there was a slight non-significant increase in mean PaCO2 (1.8 90.4 torr). This PaCO2 level was not different from the PaCO2 of saline-treated goats. The PaCO2 was successfully maintained isocapnic with respect to this apparently elevated baseline level throughout the hypoxic period. Post-methysergide, there was a small, significant decrease in pHa. This level of pHa was also significantly lower than the corresponding pHa in saline-treated goats, and remained significantly lower until 1 h of hypoxia. Methysergide administration did not alter normoxic ventilation (Fig. 1), nor did the pattern

differ from the saline-treated goats. Hypoxia significantly elevated f (Fig. 1A), VT (Fig. 1B) and V: E (Fig. 1C). There were time-dependent increases in both V: E and f during prolonged hypoxia as these values during the second and third hours of hypoxia were significantly elevated above the acute hypoxic response. Expired minute ventilation was significantly elevated above the salinetreated goats at 30 min of isocapnic hypoxia (Fig. 1C). This elevated V: E was predominately due to a significant elevation in f (Fig. 1A), with an apparent but non-significant increase in VT (Fig. 1B). No respiratory variable differed from the corresponding time point of the saline-treated animals after the first 30 min of hypoxia. V: E declined after the third hour of hypoxia and was no longer significantly different from the initial acute hypoxic response by the fourth hour of hypoxia. Upon return to room air, all ventilatory variables returned to the pre-hypoxia baseline levels and were not significantly different from the salinetreated goats. Similar to saline-treated goats, there was a significant decrease in PaCO2 after returning to room air. There were no differences in pHa, PaO2, or HCO− 3 between the pre- and post-hypoxia time points.

3.3. Post-VAH acute hypoxic response In the saline-treated goats, while the post-VAH acute hypoxic V: E was significantly elevated above the normoxic level, it was not significantly different from the pre-VAH acute hypoxic V: E (Fig. 2A). In the methysergide-treated goats, the postVAH acute hypoxic V: E was also significantly elevated above the normoxic level. However, it was slightly, but not significantly, lower than the initial acute hypoxic V: E (Fig. 2B). The acute hypoxic ventilatory response following acclimatization did not differ between saline and methysergide-treated goats.

3.4. Ketanserin Blood gas variables for the ketanserin-treated goats are summarized in Table 1. Prior to ketanserin, the PaCO2 was lower than the vehicle/ drug PaCO2 of the control and methysergide

Table 1 a Arterial pH, PCO2, PO2 and HCO− 3 before (pre-, post-drug), during (30–240) and after (room air) 4 h continous isocapnic hypoxia Post-inject

30 min

60 min

120 min

180 min

240 min

Room air

Acute 2

PaO2 (torr) Saline Methysergide Ketanserin

95.0 9 2.7 92.0 9 1.9 97.5 91.2

94.4 9 1.8 87.7 9 2.0c 97.5 9 1.2

41.7 91.0b 40.8 91.4b 40.9 90.6b

41.3 9 0.8b 41.0 9 0.5b 40.9 9 0.6b

40.1 90.6b 39.4 9 0.5b 40.3 9 1.1b

40.390.4b 39.790.5b 40.49 0.6b

40.590.6b 40.090.4b

96.292.5 93.591.8b 102.19 2.6d

40.2 9 0.5b 39.190.4b

PaCO2 (torr) Saline Methysergide Ketanserin

38.89 1.1 38.7 9 0.9 37.0 9 0.5

39.0 9 1.2 40.4 9 0.7 37.1 9 0.4c,d

39.2 9 0.8 40.5 90.7 37.0 9 0.5c,d

38.8 91.0 40.3 90.8 36.8 90.3c,d

38.9 9 0.8 40.5 90.8 37.1 9 0.5d

39.290.9 40.39 0.7 36.89 0.8c,d

39.2 91.1 40.491.0

36.2 9 1.2b 36.491.5b 38.59 1.0b 38.591.0b 33.991.0b,c,d

pHa Saline Methysergide Ketanserin

7.43 90.00 7.42 90.00b 7.429 0.01

7.42 90.00 7.399 0.01c 7.40 90.01

7.41 9 0.01 7.38 90.00c 7.41 90.01d

7.41 9 0.01 7.39 9 0.00 7.40 9 0.01

7.40 90.01b 7.39 9 0.00 7.40 9 0.01

7.399 0.01b 7.3890.01 7.40 90.01d

7.3990.01b 7.39 90.00

7.4190.01 7.40 9 0.00 7.4290.01

7.41 9 0.01 7.41 9 0.01

−1 ) HCO− 3 (mmol l Saline Methysergide Ketanserin

24.5 9 0.8 23.69 0.6 22.9 9 0.3c

24.3 9 0.9 23.0 90.5c 22.2 90.5c

23.5 90.8 22.7 9 0.6 22.3 9 0.5

23.4 9 0.7 23.1 90.6 22.0 90.5c

23.1 9 0.6 22.9 9 0.5 22.0 9 0.7

22.290.6b 22.3 90.6 21.99 0.7

22.590.7b 23.1 9 0.5

22.090.7b 22.6 9 0.5 21.190.6d

22.190.9b 23.2 9 0.5

a

Blood gas variables during Significantly different from c Significantly different from d Significantly different from b

the post-acclimatization acute hypoxic response are represented under Acute 2. baseline post drug (PB0.05). corresponding time point in control goats. corresponding time point in the methysergide-treated goats. Values are means 9SE.

J.K. Herman et al. / Respiration Physiology 118 (1999) 25–37

Pre-Inject

29

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J.K. Herman et al. / Respiration Physiology 118 (1999) 25–37

Fig. 1. Ventilatory variables following saline ( ) or methysergide (D) administration during 4 h of sustained isocapnic hypoxia. (A) Respiratory frequency (f). (B) Tidal volume (VT) and (C) expired minute ventilation (V: E). Time (0), normoxic control ventilation 30 min post-drug administration. Time (270), return to normoxia. * Significantly different from control post drug (PB 0.05). ** Significantly increased from acute hypoxic ventilatory response (PB 0.05). c Significantly different from corresponding time point in control goats (PB 0.05). Values are means 9 SE. Corresponding blood gas data are shown in Table 1.

J.K. Herman et al. / Respiration Physiology 118 (1999) 25–37

groups. Similarly, the HCO− 3 concentration was significantly lower than that of the control goats. Ketanserin did not alter any of the blood gas variables (Table 1). Following ketanserin administration, there were non-significant decreases in f and increases in VT resulting in no change in V: E

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(Fig. 3). Expired minute ventilation increased significantly during hypoxia and reached a level similar to the saline-treated goats (Fig. 3C). The elevated V: E was achieved by elevations in both f and VT (Fig. 3A and B, respectively). The acute hypoxic V: E was significantly lower than when the

Fig. 2. Ventilatory response to acute isocapnic hypoxia before (open symbols), and 30 – 60 min after (closed symbols) the completion of 4 h isocapnic hypoxia for saline (A) and methysergide (B) treated goats. Values are means 9 SE. Corresponding blood gas data are shown in Table 1.

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Fig. 3. Ventilatory variables following saline ( ) or ketanserin ( ) administration during 3 h of sustained isocapnic hypoxia. (A) Respiratory frequency (f). (B) Tidal volume (VT). and (C) expired minute ventilation (V: E). Time (0), normoxic control ventilation 30 min post-drug administration. Time (240), return to normoxia. * Significantly different from baseline post drug (PB 0.05). ** Significantly increased from acute hypoxic ventilatory response (PB0.05). c Significantly different from corresponding time point in saline goats (P B 0.05). Saline response is as depicted in Fig. 1, saline response plotted again with ketanserin for comparison. Values are means 9 SE. Corresponding blood gas data are shown in Table 1.

goats were given methysergide. Expired minute ventilation increased in a time-dependent manner with continuous hypoxia. The second and third

hours of hypoxia were significantly elevated above the initial hypoxic ventilatory response. At these time points, the ketanserin-treated group did not

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differ from the control or methysergide-treated goats. Following the return to room air, all ventilatory variables (Fig. 3), pHa and PaO2 returned to the pre-hypoxic control levels while PaCO2 decreased significantly (3.190.8 torr) from baseline (PB 0.05; Table 1).

3.5. Cardio6ascular response Heart rate (fH) and mean arterial pressure (MAP) for the saline, methysergide and ketanserin-treated goats are summarized in Table 2. Initially, there were no differences in fH between goats in any treatment group. Ketanserin induced a significant increase in fH; the post-ketanserin fH also differed from the saline, and methysergide-treated goats. Hypoxia induced a significant increase in fH for the saline and methysergidetreated goats, but not in the ketanserin-treated animals. During hypoxia, there was no difference in fH between any group nor did fH change with continuous hypoxia. Methysergide induced a significant increase in MAP but did not differ from the saline or ketanserin-treated goats prior to hypoxia. Hypoxia did not induce any systematic change in blood pressure.

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4. Discussion The purpose of the present study was to examine the role of 5-HT receptor activation on the acute hypoxic ventilatory response and VAH. Our data show that, while methysergide augmented the acute hypoxic ventilatory response, it did not result in any qualitative difference in the time-dependent increase in ventilation associated with sustained isocapnic hypoxia. The results of the ketanserin study suggest that this augmented acute hypoxic V: E is not dependent on 5-HT2A/2C receptor activation. A third finding of this study, which may not be related to 5-HT, is that there was a significant fall in PaCO2 below baseline on return to normoxia following sustained isocapnic hypoxia. This observation is consistent with a marginal degree of ventilatory de-acclimatization or long-term facilitation (Bisgard and Neubauer, 1995; Powell et al., 1998). Systemic depletion of 5-HT by PCPA induces hyperventilation, resulting in a prolonged decrease in PaCO2 (Mitchell et al., 1983; Olson, 1987). Neither methysergide nor ketanserin produced similar effects on ventilation during normoxia in the present study. Unlike what was found with methysergide, 5-HT depletion (Olson, 1987) did not result in an

Table 2 Heart rate (fH; beats min−1), and mean arterial pressure (MAP; mmHg) before (pre-, post-drug), during (30–240) and after (room air) 4 h continous isocapnic hypoxiaa Pre-drug

Post-drug

30 min

60 min

120 min

180 min

240 min

Room air

Acute 2

fH Saline Methysergide Ketanserin

89 910 72 95 68 93b

859 10 789 8 1129 11c,d

1269 14b 1379 10b 123 9 11

1219 8b 1339 5b 1209 7

137 93b 142 93b 126 910

139 95b 146 97b 125 9 9

141 97b 133 95b

95 9 7 87 9 8 76 9 4b

130 9 4b 128 98b

MAP Saline Methysergide Ketanserin

117 95 111 93b 116 95

118 9 6 1289 4 1109 7d

1169 5 1239 4 1139 6

115 95 122 94 1139 5

118 9 4 122 9 5 113 99

120 93 124 96 118 9 7

117 93 125 97

a

116 95 120 97 114 95

118 9 4 124 97

Cardiovascular variables during the post-acclimatization acute hypoxic response are represented under Acute 2. Significantly different from baseline post drug (PB0.05). c Significantly different from corresponding time point in control goats. d Significantly different from corresponding time point in the methysergide-treated goats. Values are means 9SE. Corresponding blood gas data are shown in Table 1. b

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augmentation of the acute hypoxic ventilatory response. However, similar to what was reported with 5-HT depletion (Olson, 1987), 5-HT receptor blockade did not alter the time-dependent increase in V: E associated with VAH. Interestingly, in both the Olson (1987) study, and following methysergide in the present study, the difference in V: E between the control and treated groups during hypoxia was consistent throughout most of the protocol. The fall in V: E at the fourth hour of hypoxia may have been due to methysergide decreasing below an effective concentration at this time point due to its relatively short half-life ( : 45–60 min; Bredberg et al., 1986; Silberstein, 1998). This short half-life makes it difficult to firmly define a role for 5-HT during VAH. Indeed, the augmentation of the acute hypoxic response following methysergide would suggest that 5-HT is involved in the modulation of ventilatory magnitude during hypoxia. This modulation was sufficient to elevate the acute V: E to a level in which it did not differ from the fully acclimatized V: E of the saline-treated goats. The V: E at the second and third hour of hypoxia in the methysergide protocol was significantly elevated above the acute V: E, suggesting that 5-HT does not significantly contribute to the time-dependent component of VAH. However, because of the relatively short half-life of methysergide, at this time we cannot make any firm conclusions about the significance of 5-HT during VAH. One potential criticism of this study is that the observed ventilatory changes were due to the small decrease in pH that occurred following methysergide (Table 1). To test this possibility, the acute hypoxic response was examined in two goats used in this study while their PaCO2 was elevated 2 and 4 torr above baseline. While these changes induced increases in the normoxic-breathing pattern, only the 4 torr PaCO2 increase induced an acute hypoxic V: E of a similar magnitude to that observed with methysergide. However, as the normoxic/hypercapnic baseline V: E was also significantly elevated, the change in V: E associated with hypercapnic-hypoxia was less (: 10 vs. 30 l min − 1 with methysergide). Therefore, we are confident that the acute hypoxic V: E that was observed was larger in magnitude than could be

explained by the small changes in PaCO2 and pHa following methysergide administration. While the same goats were used in all experiments, the pre-ketanserin baseline PaCO2 and HCO− levels were significantly lower than the 3 pre-saline levels. However, despite the lower PaCO2 and HCO− 3 levels, the ketanserin and saline-treated goats had a similar pHa and were similar in both magnitude and time-course of VAH. We chose to maintain this lower PaCO2 to avoid inducing a relative hypercapnic stimulus. Therefore, while the results suggest that 5-HT2A/2C receptor subtypes do not play a role in modulating the acute hypoxic response, the differences in baseline PaCO2 make it difficult to completely rule out their involvement. It may also be argued that methysergide and ketanserin did not bind to and antagonize the same populations of 5-HT2 receptor subtypes and therefore did not elicit similar hypoxic responses. Methysergide has a slightly higher affinity for the 5-HT2C over the 5-HT2A receptor subtype while ketanserin has the opposite affinity differences (Hoyer et al., 1994). Therefore, it is possible that the concentration of ketanserin used was not sufficient to induce adequate blockade of the appropriate receptor population. However, the dose of ketanserin that was used is similar to the highest clinical dose (40 mg, twice daily) used in humans to effectively treat hypertension (Persson et al., 1991) and is larger than the reported dose that is effective at reducing post-anesthetic shivering in humans (10 mg; Crisinel et al., 1997). Additionally, ketanserin elicited consistent, albeit small decreases in f and increases in VT during normoxia, and similar VT levels throughout hypoxia as the methysergidetreated goats, thereby suggesting that sufficient ketanserin was available and that these two antagonists were acting at similar receptor populations. Serotonin2A receptors have been reported in the goat carotid body (Wang et al., 1998) and the 5-HT2A/2C receptor subtypes contribute to the 5HT induced inhibition of the cat carotid body (Kirby and McQueen, 1984). Preliminary results, from our laboratory, examining single fiber recordings from the goat carotid body indicate that 5-HT induces similar effects as that reported by Kirby and McQueen (1984). The similar in-

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creases in VT during hypoxia in the methysergide and ketanserin-treated goats may reflect, in part, a similar degree of blockade of the 5-HT2A receptor within the carotid body. Ketanserin has a longer elimination half-life (Michiels et al., 1988) than that reported for methysergide (Bredberg et al., 1986; Silberstein, 1998). Therefore, while the two drugs were administered in molar equivalent concentrations, the ketanserin concentration would have been higher than the methysergide concentration when the acute hypoxic V: E was recorded (: 1 h post drug). Therefore, if the methysergide induced hypoxic response was mediated via blockade of 5-HT2A/2C receptor subtypes, it should also have been evident with the dose of ketanserin that was used. Finally, since this dose of ketanserin nearly doubled heart rate (not present with methysergide), we were reluctant to increase the dose further. In addition to being a broad-spectrum 5-HT receptor antagonist, methysergide has also been reported to be a partial to full 5-HT1 receptor agonist (Hoyer et al., 1994; Silberstein, 1998). Therefore, a portion of the hypoxic response induced by methysergide administration may have been mediated via 5-HT1 stimulation rather than 5-HT2A/2C blockade. Systemic administration of 5-HT1A agonists have been shown to induce increases in respiratory rate by decreasing inspiratory duration (Lalley et al., 1994; Wilken et al., 1997). This is consistent with the observation that, following methysergide, the increase in V: E during hypoxia was primarily due to an increase in f rather that VT. The 5-HT1A receptor does not appear to be present in the carotid body (Dashwood et al., 1990), which would suggest that if the methysergide response is elicited through this 5-HT receptor subtype, it is not carotid body mediated. Preliminary observations in our laboratory, using the 5-HT1A agonist, 8-OH DPAT, have indicated that stimulation of the 5-HT1A receptor induces large increases in f without changing VT. However, while this finding is suggestive, we cannot rule out the possibility that methysergide was acting at either a different 5-HT receptor or through multiple 5-HT receptor subtypes (see Hoyer et al., 1994). In previous studies from our laboratory, the V: E response to acute hypoxia has been consistently elevated following VAH (Ryan et al., 1993;

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Dwinell et al., 1997a; Janssen et al., 1998). In the present study, this effect was not clearly shown. This discrepancy may be explained by the fact that PaCO2 decreased post-VAH in this study and, thus, the subsequent assessment of the acute hypoxic response was run at this lower PaCO2. Isocapnic hypoxia significantly increases the slope of both the whole animal (Engwall and Bisgard, 1990) and carotid body CO2 response curve (Dwinell et al., 1997b). To avoid potential complications of an increase in CO2 responsiveness, we assessed hypoxic sensitivity using the new baseline PaCO2. Therefore, the post-VAH acute hypoxic response may have been diminished somewhat. In the saline-treated goats, although there was no difference between the initial and post-VAH acute hypoxic response, the magnitude of the post-VAH response was similar to that previously reported. Therefore, our results suggest that there was a slight increase in hypoxic sensitivity following VAH. The post-VAH acute hypoxic response was similar in the methysergide and saline-treated goats, indicating that there was either insufficient methysergide remaining, or that the increase in oxygen sensitivity associated with VAH is not 5-HT dependent. Previous studies using this protocol to induce VAH found either no change (Engwall and Bisgard, 1990; Ryan et al., 1993; Dwinell et al., 1997a), or a small but significant fall in PaCO2 following isocapnic VAH (Janssen et al., 1998). In the present study, the fall in PaCO2 following isocapnic VAH was larger than previously reported. A major difference in protocol between this and previous studies is that previous studies used unilaterally carotid body denervated goats whereas both carotid bodies were intact in the present study. While this protocol difference did not result in any alterations in the time course of VAH, the magnitude of hypoxic V: E was larger than previously reported, an effect that may have contributed to the hyperventilation post-VAH. The resultant decrease in PaCO2 was stable, and persisted for the duration that the animals were maintained in normoxia (30–60 min). It is unknown if this fall in PaCO2 is related to the de-acclimatization observed following poikilocapnic hypoxia in goats (Engwall and Bisgard, 1990).

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The apparent fall in PaCO2 following continuous isocapnic hypoxia suggests a similarity between VAH and some other reported hypoxic ventilatory responses. Episodic stimulation of the carotid body (or carotid sinus nerve) leads to a prolongation of respiratory output after the stimulus at the CB is removed (Millhorn et al., 1980). Such long term facilitation (LTF) can be elicited in awake goats and, thus, supplemental CO2 is necessary to maintain isocapnic conditions following the stimulus protocol (Turner and Mitchell, 1997). In contrast, continuous isocapnic hypoxia of a similar cumulative duration (30 min) or following 4 h does not induce LTF in awake goats (Dwinell et al., 1997a). However, PaCO2 appeared to decrease slightly following hypoxia in the study of Dwinell et al. (1997a). Had isocapnia been maintained, a similar LTF of ventilation may have been elicited to that reported by Turner and Mitchell (1997). Serotonin receptor activation has been reported to be necessary for LTF, as it is eliminated by methysergide (Millhorn et al., 1980; Bach and Mitchell, 1996) or ketanserin administration (Kinkead and Mitchell, 1999). Similar to the saline-treated goats, a significant fall in PaCO2 following VAH occurred in both the ketanserin and methysergide-treated animals. While these results suggest that 5-HT receptor activation is not necessary for the manifestation of this fall in PaCO2, the long time course of these experiments makes it difficult to ascertain whether effective concentrations of either drug remained. In summary, we used methysergide and ketanserin to investigate the role of 5-HT receptor activation in the awake, acute hypoxic ventilatory response and VAH. We have shown that methysergide augments the ventilatory response to acute hypoxia suggesting that 5-HT modulates the initial hypoxic ventilatory response. We also showed that this modulation does not appear to be acting through the 5-HT2A/2C receptor subtype. Similar to previous reports (Olson, 1987), we showed that 5-HT does not appear to be necessary for VAH since the time course of V: E increase between the control, methysergide and ketanserin-treated goats during VAH did not differ. Finally, we have observed an apparent hyperventilation following isocapnic VAH in awake goats, suggesting that 4

h of continuous hypoxia may elicit a post-hypoxia facilitation (de-acclimatization or LTF) of ventilation; the significance of this latter effect remains to be investigated.

Acknowledgements We Josue tance. 07654

would like to thank Gordon Johnson and Pizarro for their excellent technical assis(Supported by NIH Grants HL 15473, HL and HL 10069).

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