AJP - Regu Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 295: R1599-R1612, 2008. First published September 10, 2008; doi:10.1152/ajpregu.00020.2008
0363-6119/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/R1599    most recent
00020.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, S. M.
Right arrow Articles by Wiegel, L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, S. M.
Right arrow Articles by Wiegel, L. M.

EXERCISE AND RESPIRATORY PHYSIOLOGY

Inhibitory and excitatory effects of µ-, {delta}-, and {kappa}-opioid receptor activation on breathing in awake turtles, Trachemys scripta

Stephen M. Johnson, Matthew E. Kinney, and Liana M. Wiegel

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin

Submitted 13 August 2008 ; accepted in final form 3 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
For ectothermic vertebrates, such as reptiles, the effects of opioid receptor subtype activation on breathing are poorly understood. On the basis of previous studies on mammals and lampreys, we hypothesized that µ- and {delta}-opioid receptor (MOR and DOR, respectively) activation would cause respiratory depression, whereas {kappa}-opioid receptor (KOR) activation would have no effect. To address this question, we measured respiration in awake, freely swimming adult red-eared slider turtles (Trachemys scripta) before and after injection with agonists for specific opioid receptors. Injection of the MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin acetate salt (DAMGO, 1.5 or 6.5 mg/kg) decreased ventilation (VE) by 72 ± 9% and 95 ± 3%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in tidal volume (VT). DOR agonists, such as [D-Pen2,5]-enkephalin hydrate (DPDPE, 5.0 mg/kg) and [D-Ala2,D-Leu5]-enkephalin acetate salt (DADLE, 6.3 mg/kg), decreased VE by 44 ± 10% and 89 ± 4%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in VT. DADLE also increased breath duration by a maximum of 25 ± 9% at 6.0 h after injection. The KOR agonist U-50488 (6.2 mg/kg) increased VT by a maximum of 52 ± 30% at 5.0 h after injection, with variable nonsignificant changes in VE and breathing frequency. Naloxone injections (0.25–0.5 mg/kg) 1.0 h before opioid agonist injections blocked all DAMGO-dependent effects, DPDPE-dependent frequency depression, and DADLE-dependent breath duration augmentation for 2.0 h after agonist injections. These results show that MOR and DOR activation causes respiratory depression as a result of decreased breathing frequency, whereas VT is increased after KOR activation.

reptile; respiration; chelonian

OPIOID DRUGS ARE COMMONLY administered in human and veterinary medicine to relieve pain, as well as reduce coughing, diarrhea, and anxiety. However, opioid drugs have deleterious side effects, such as respiratory depression, hypotension, nausea and vomiting, constipation, drowsiness, and physical dependence (18, 38). In mammals, opioid drugs can produce excitatory or inhibitory effects on breathing, depending on the drug, route of administration, presence of anesthesia, and species (42, 44). Even when specific opioid receptor agonists are microinjected into different parts of the ventral respiratory group (VRG) in the medulla of anesthetized rats, there are a variety of time-dependent changes in cardiorespiratory function, depending on the precise location of the injections (25, 26). Thus, understanding how opioid drugs alter respiration is important for development of effective drugs with fewer unwanted side effects.

In ectothermic vertebrates, very little is known with respect to how opioid drugs modulate breathing. In intact frogs, morphine, a µ-, {delta}-, and {kappa}-opioid receptor (MOR, DOR, and KOR, respectively) agonist, reduces lung ventilation frequency, lung episode frequency, and the number of lung breaths per episode (51, 52). Similar results were obtained when the MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin acetate salt (DAMGO) was applied to isolated brain stems from the same species, suggesting that these effects were primarily due to central MOR activation (51, 52). In isolated lamprey brain stems, DAMGO and the DOR agonist [D-Pen2,5]-enkephalin hydrate (DPDPE) reduce respiratory burst frequency without altering burst pattern, whereas the KOR agonist U-50488 had no effect (33). In awake red-eared slider turtles (Trachemys scripta), morphine and butorphanol (a mixed KOR agonist and MOR agonist/antagonist) depress ventilation as a result of a 60–80% reduction in breathing frequency (45). Thus MOR activation appears to decrease ventilation (VE) in frogs, lampreys, and turtles as a result of decreased breathing frequency, but the effects of DOR and KOR activation on VE and breathing pattern are not as well understood in ectothermic vertebrates. In turtles, we hypothesized that MOR and DOR (but not KOR) activation would produce respiratory depression.

To address this question, breathing was measured in awake, freely swimming turtles that were placed in a water-filled tank equipped with a small inverted air-filled chamber for breathing. A pneumotachograph was used to measure VE, breathing frequency, tidal volume (VT), and the number of breaths per episode. Agonists for MOR, DOR, and KOR receptors were administered, and changes in breathing were measured up to 6.0 h after injection. The main findings were that MOR and DOR activation depressed VE by decreasing breathing frequency, whereas KOR activation increased VT.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures were approved by the Animal Care and Use Committee at the University of Wisconsin-Madison School of Veterinary Medicine. Adult red-eared slider turtles (T. scripta, n = 16 male and 16 female, 805 ± 14 g body wt) were obtained from commercial suppliers and kept in a large open tank, where they had access to water for swimming and to heat lamps and dry areas for basking. Room temperature was set to 27–28°C with light for 14 h/day. Turtles were fed ReptoMin floating food sticks (Tetra, Blacksburg, VA) 3–4 times/wk.

Turtle VE Measurements

VE in awake freely swimming turtles was measured using established methods (9). Turtles were placed in a plastic container (16 x 42 x 42 cm) filled to the top with water at room temperature (Fig. 1A). An 8.0-cm-diameter circle was cut in the top, and a 250-ml plastic container was inverted and sealed over the hole. This inverted plastic "breathing" chamber provided the only location within the tank where the turtles could breathe. Flowmeters maintained gas flow (room air) into the chamber at ~500 ml/min. A pneumotachograph (Godart, Gould Electronics, Eastlake, OH) was attached to the breathing chamber exit hole for measurement of airflow (Fig. 1A). After 1 day of conditioning (e.g., 4–6 h in the chamber), turtles were placed in the chamber to breathe room air for 2 h to establish baseline VE, defined as the 60-min period before drug or saline injection. Turtles were removed from the tank, injected subcutaneously with drugs, and returned to the tank to breathe room air for another 3–6 h. A minimum of 2 wk were allowed between experiments. The same turtles (or turtles obtained at the same time of year) were used for the saline and drug injection studies to minimize potentially confounding batch or seasonal effects.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. A: schematic drawing of a turtle in a water-filled tank (left) that allows breathing in an inverted plastic chamber connected to a pneumotachograph. Turtles move their pectoral and pelvic girdles back and forth in a bellows-like manner to decrease (expiration) and increase (inspiration) lung volume (note arrows and shaded area). A voltage trace from a pneumotachograph (right) shows upward deflections as expiration and downward deflections as inspiration. B: baseline breathing (left) during the 1.0-h period before injection of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin acetate salt (DAMGO) at 6.5 mg/kg. At 4.5 h after injection (right), breathing frequency was decreased, with little change in tidal volume (VT). Horizontal bars below traces indicate duration of discrete breathing episodes.

 
Electrical signals from the pneumotachograph were saved to a computer using a data acquisition system (LabPro, Vernier Software & Technology, Beaverton, OR) and analyzed offline using Clampfit software (Axon Instruments, Union City, CA). Expiratory and inspiratory signals primarily occurred in pairs, with expiration preceding inspiration (Fig. 1A). For calculation of VE (ml·min–1·kg–1), the area under individual expiratory traces was measured and the sum of areas within a given time period was determined. Breathing frequency was defined as the average number of breaths per minute. VT (ml/kg) was calculated as VE ÷ breathing frequency. Breath duration was defined as the time for completion of one expiratory/inspiratory cycle. A breath was considered part of an episode if the time interval between two breaths was less than the average breath duration (Fig. 1B).

Pneumotachograph Calibration

The pneumotachograph was calibrated according to published methods (9). One end of a section of plastic tubing (0.3 mm ID, ~40 cm long) was inserted into the breathing chamber, and the other end was connected to a motor-driven 25-ml glass syringe. The syringe was set at different volumes (range 4.5–22.5 ml) and rhythmically moved back and forth at cycle periods of 1.5–4.5 s (similar to the duration of 1 turtle expiratory-inspiratory cycle). For a given syringe volume, expiratory trace areas (Fig. 1A) were averaged at different frequencies and plotted vs. the logarithm of the syringe frequency. These plots showed that expiratory area measurements were relatively insensitive to frequency, with a maximum of 10–20% error in VE and VT measurements only at the highest VT and frequencies in turtles. Since baseline breathing measurements in the present study were similar to those previously reported (9, 19, 20, 30) and most drugs decreased breathing frequency and increased breath duration, the impact of systematic pneumotachograph errors was deemed to be minimal with respect to the major findings.

Drugs

DAMGO, DPDPE, the DOR agonist [D-Ala2,D-Leu5]-enkephalin acetate salt (DADLE), and (–)-trans-(1S,2S)-U-50488 hydrochloride hydrate (U-50488) were purchased from Sigma-Aldrich (St. Louis, MO). Naloxone (0.4 mg/ml solution), an MOR, DOR, and KOR antagonist, was purchased from Hospira (Lake Forest, IL).

Data Analysis

Since a crossover experimental design was used for the DAMGO, DPDPE, and DADLE studies, a two-way repeated-measures ANOVA was used to analyze the data (Sigma Stat, Jandel Scientific Software, San Rafael, CA). Since a between-subjects experimental design was used for all other respiratory data, a two-way ANOVA was used to analyze these data. If the normality or equal variance assumptions were not satisfied, data were ranked, and the ANOVA was performed on ranked data. Post hoc comparisons were made using the Student-Newman-Keuls test. Baseline and postinjection expiratory and inspiratory durations were compared using t-tests. Values are means ± SE. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Respiratory Effects of MOR Activation

Saline injections (n = 7) did not alter VE, breathing frequency, VT, breath duration, or breaths per episode (Fig. 2), except for a minor significant increase in VE and breathing frequency 1.0 h after injection (Fig. 2, A and B) that was probably due to handling during the injection. In the same turtles, VE was rapidly reduced from 16.4 ± 1.3 to 4.3 ± 0.4 ml·min–1·kg–1 by DAMGO at 1.5 mg/kg and from 16.4 ± 3.2 to 1.0 ± 0.7 ml·min–1·kg–1 by DAMGO at 6.5 mg/kg at 4.0 h after injection (P < 0.001; Figs. 1B and 2A). The decrease in VE was due to nearly identical changes in breathing frequency (Fig. 2B) with no change in VT (Fig. 2C). Breathing frequency decreased 73% (1.5 mg/kg) and 93% (6.5 mg/kg) from baseline 4.0 h after injection (P < 0.001; Fig. 2B). DAMGO at 1.5 mg/kg produced a time-dependent increase in breath duration from 2.73 ± 0.20 s (baseline) to 3.26 ± 0.16 s at 6.0 h after injection (P < 0.018; Fig. 2D). Expiratory duration was unchanged (1.29 ± 0.10 s at 0.0 h vs. 1.46 ± 0.08 s at 7.0 h, P = 0.59), whereas inspiratory duration increased from 1.44 ± 0.11 s (0.0 h) to 1.80 ± 0.10 s at 7.0 h (P = 0.03). In contrast, DAMGO (6.5 mg/kg) did not increase breath duration; the significant drug effect (P = 0.009) was likely due to large differences in baseline breath duration. Breaths per episode decreased from 4.1 ± 0.5 (baseline) to 3.1 ± 0.6 after injection of DAMGO at 1.5 mg/kg and from 3.4 ± 0.5 (baseline) to 1.6 ± 0.1 breaths/episode after injection of DAMGO at 6.5 mg/kg at 6.0 h after injection (P < 0.001 for drug effect; Fig. 2E). In separate experiments, injection of naloxone (0.25 mg/kg) 1.0 h before saline (n = 7) or DAMGO (1.5 mg/kg, n = 8) resulted in no drug- or time-dependent changes in VE, breathing frequency, VT, breath duration, or breaths per episode (Fig. 3). Data analysis following opioid agonist injection was restricted to 2.0 h after injection with the agonist, because separate pilot studies showed that this naloxone dose completely reversed morphine-induced respiratory depression for 3.0 h following the naloxone injection (unpublished observations).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. DAMGO decreases ventilation (VE), breathing frequency, and breaths per episode. A: VE decreased within 2.0 h and remained depressed for up to 6.0 h after injection with DAMGO at 1.5 or 6.5 mg/kg. B: breathing frequency decreased significantly in an identical manner to VE. C: VT was unaltered by DAMGO. D: DAMGO at 1.5 mg/kg increased breath duration in a time-dependent manner compared with saline. A significant drug effect was observed for DAMGO at 6.5 mg/kg, but this effect was due to a higher baseline value at time 0. E: DAMGO at 1.5 and 6.5 mg/kg significantly decreased number of breaths per episode. Values are means ± SE. *P < 0.05 vs. saline. #P < 0.05 vs. baseline. {dagger}P < 0.05 for drug effect.

 

Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Naloxone blocks effects of DAMGO on breathing. Naloxone (0.25 mg/kg) was injected at arrow 1 (immediately after baseline data were obtained), and saline or DAMGO (1.5 mg/kg) was injected at arrow 2 (immediately after 1.0-h exposure to naloxone). VE (A), breathing frequency (B), VT (C), breath duration (D), and breaths per episode (E) did not differ between turtles injected with naloxone-saline and those injected with naloxone-DAMGO.

 
Respiratory Effects of DOR Activation

DPDPE and DADLE were tested, because they are commonly used DOR agonist drugs and because their specificity and pharmacodynamics are not well understood in turtles. In saline-injected turtles (n = 10), there were no changes in VE, breathing frequency, VT, breath duration, or breaths per episode (see Fig. 5), except for a large increase in breaths per episode 5–6 h after injection in a single turtle breathing in several long episodes (P = 0.046 for drug effect; see Fig. 5E). In the same turtles, DPDPE (5 mg/kg) decreased VE from 16.6 ± 2.4 (baseline) to 9.6 ± 2.0 ml·min–1·kg–1 at 6.0 h after injection (P < 0.001 for drug effect; Figs. 4A and 5A ) as a result of a decrease of breathing frequency from 2.1 ± 0.4 (baseline) to 1.2 ± 0.3 breaths/min (P = 0.002 for drug effect; Fig. 5B). VT, breath duration, and breaths per minute were unchanged by DPDPE (Fig. 5, C–E). In separate experiments, when naloxone (0.5 mg/kg) was injected 1.0 h before DPDPE injections (5 mg/kg, n = 6), DPDPE decreased VE from 22.5 ± 2.5 (1.0 h time point in Fig. 6A) to 14.8 ± 3.9 ml·min–1·kg–1 2.0 h later (P = 0.032 for drug effect; Fig. 6A), but there were no DPDPE-dependent changes in breathing frequency, VT, breath duration, or breaths per episode (Fig. 6, B–E). Thus DPDPE-dependent changes in breathing frequency and breaths per episode, but not VE, were blocked by naloxone.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. DPDPE decreases VE and breathing frequency. A: VE decreased within 2.0 h and remained depressed for up to 6.0 h after DPDPE injection (5.0 mg/kg) compared with saline injections. B: breathing frequency decreased in parallel to VE. C and D: VT and breath duration were unaltered by DPDPE. E: DPDPE did not alter breaths per episode; significant drug effect was due to large variability in saline-injected turtles. *P < 0.05 vs. saline. #P < 0.05 vs. baseline. {dagger}P < 0.05 for drug effect.

 

Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. [D-Pen2,5]-enkephalin hydrate (DPDPE, 5.0 mg/kg), [D-Ala2,D-Leu5]-enkephalin acetate salt (DADLE), and U-50488 alter breathing. A: baseline breathing traces (left) before DPDPE injection (5 mg/kg). Note decrease in breathing frequency at 5.5 h after injection with DPDPE (right). B: at 4.5 h after injection with DADLE (6.3 mg/kg), VE decreased due to decreased breathing frequency. C: at 4.5 h after injection with U-50488 (6.2 mg/kg), VT was increased.

 

Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 6. Naloxone blocks effects of DPDPE on breathing frequency, but not VE. Naloxone (0.5 mg/kg) was injected at arrow 1 (immediately after baseline data were obtained), and saline or DPDPE (5.0 mg/kg) was injected at arrow 2 (immediately after 1.0-h exposure to naloxone). A: despite naloxone pretreatment, DPDPE decreased VE. B: DPDPE-dependent decrease in breathing frequency was abolished. C and D: there were no time-dependent effects on VT or breath duration. E: significant drug effects on breaths per episode were due to differences in baseline data at time 0. {dagger}P < 0.05 for drug effect.

 
Similarly, DADLE (6.3 mg/kg, n = 8) decreased VE as a result of a decrease in frequency (Figs. 4B and 7). For the same turtles injected with saline, VE started at 17.7 ± 2.5 ml·min–1·kg–1, increased to 28.8 ± 4.1 ml·min–1·kg–1 at 1.0 h after injection (P = 0.034; likely due to handling during the injection), and returned to near-baseline levels for the next 5.0 h (Fig. 7A). DADLE decreased VE from 17.7 ± 3.6 (baseline) to 2.3 ± 0.8 ml·min–1·kg–1 at 6.0 h after injection (P < 0.001 for drug effect; Fig. 7A). DADLE decreased breathing frequency from 0.9 ± 0.2 (baseline) to 0.1 ± 0.1 breaths/min at 6.0 h after injection (P < 0.001 for drug effect; Fig. 7B). VT was unchanged in saline- and DADLE-injected turtles (P = 0.207; Fig. 7C). Breath duration in DADLE-injected turtles increased steadily with time from 2.67 ± 0.20 (baseline) to 3.34 ± 0.24 s at 6.0 h (P < 0.001 for drug effect; Fig. 7D). Expiratory duration was unchanged (1.22 ± 0.09 s at 0.0 h and 1.31 ± 0.12 s at 7.0 h, P = 0.49) whereas inspiratory duration increased from 1.48 ± 0.13 s at 0.0 h to 2.03 ± 0.22 s at 7.0 h (P = 0.03). In saline-injected turtles, breaths per episode increased from 5.2 ± 0.8 (baseline) to a maximum of 8.0 ± 1.6 at 1.0 h after injection (P = 0.012) but then returned to near-baseline levels 3–6 h after injection (Fig. 7E). DADLE decreased breaths per episode from 5.0 ± 1.3 (baseline) to 1.7 ± 0.1 at 6.0 h after injection (P < 0.001 for drug effect; Fig. 7E).


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. DADLE decreases VE, breathing frequency, and breaths per episode. A: VE decreased within 2.0 h and remained depressed for up to 6.0 h after injection with DADLE (6.3 mg/kg) compared with saline. B: breathing frequency decreased significantly in an identical manner to VE. C: VT was unaltered by DADLE. D: breath duration increased in a time-dependent manner following the DADLE injections. E: DADLE significantly decreased the number of breaths per episode within 2.0 h. *P < 0.05 vs. saline. #P < 0.05 vs. baseline. {dagger}P < 0.05 for drug effect.

 
In separate experiments, when naloxone (0.25 mg/kg) was injected 1.0 h before DADLE injections (6.3 mg/kg, n = 5), most of the respiratory effects of DADLE were not attenuated. For example, with naloxone pretreatment, VE decreased from 29.0 ± 6.5 (baseline) to 13.3 ± 4.6 ml·min–1·kg–1 at 2.0 h after injection with DADLE (P = 0.037 for drug effect; Fig. 8A), and breathing frequency decreased from 1.6 ± 0.2 (baseline) to 0.8 ± 0.2 breaths/min at 2.0 h after injection with DADLE (P = 0.041; Fig. 8B). VT, breath duration, and breaths per episode were not altered by naloxone-saline or naloxone-DADLE injections (Fig. 8, C–E). Thus naloxone only blocked the DADLE-dependent decrease in bursts per episode.


Figure 8
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 8. Naloxone fails to block most of DADLE's effects on breathing. Naloxone (0.25 mg/kg) was injected at arrow 1 (immediately after baseline data were obtained), and saline or DADLE (6.3 mg/kg) was injected at arrow 2 (after 1.0-h exposure to naloxone). A: VE decreased with time following DADLE injection with naloxone pretreatment. B: breathing frequency also decreased with time in a manner similar to VE. There was no change in VT (C) or breaths per episode (E), but DADLE-dependent increase in breath duration was blocked (D). {dagger}P < 0.05 for drug effect.

 
Respiratory Effects of KOR Activation

Compared with saline (n = 8), U-50488 injections (6.2 mg/kg; n = 7) did not alter VE (P = 0.489), despite highly variable effects and a trend toward increased VE 4–5 h after injection (Figs. 4C and 9A). During the postinjection period, one turtle stopped breathing for 3.0 h, while VE was increased above 33 ml·min–1·kg–1 in three turtles. Similarly, breathing frequency was not altered (P = 0.073), despite the high variability (Fig. 9B). VT, however, increased from 11.0 ± 1.4 ml/kg (baseline) to a maximum of 16.7 ± 3.3 ml/kg at 5.0 h after injection (P < 0.01 for drug effect; Fig. 9C). Breath duration was unaltered by U-50488 compared with saline injections (P = 0.90; Fig. 9D). For U-50488-injected turtles, breaths per episode were highly variable, with an initial decrease 2.0 h after injection followed by a gradual increase (P = 0.019 for drug-time interaction; Fig. 9E). The statistical power for all comparisons except VT was <0.80.


Figure 9
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 9. U-50488 increases VT. A: compared with saline injections, VE remained relatively constant for 1–3 h after injection with U-50488 (6.2 mg/kg) before VE became highly variable. B: breathing frequency showed considerable variability after injection, but changes were not significant. C: VT increased steadily within 1–3 h after injection to a level that lasted 3–6 h after injection. D: breath duration was not altered by U-50488. E: number of breaths per episode was significantly reduced 2.0 h after injection but then increased above baseline and saline levels 6.0 h after injection. *P < 0.05 vs. saline. #P < 0.05 vs. baseline. {dagger}P < 0.05 for drug effect.

 
A comparison of breathing in naloxone-saline- (n = 7) and naloxone-U-50488-injected turtles (n = 6, 6.2 mg/kg) showed no differences in VE, breathing frequency (significant drug effects due to different baseline values), and breath duration (Fig. 10, A, B, and D). There was a time-dependent 20% increase in VT in naloxone-U-50488-injected turtles (P = 0.027; Fig. 10C). In naloxone-U-50488-injected turtles, breaths per episode decreased from 4.7 ± 1.1 (baseline) to 3.0 ± 0.4 breaths/min (P = 0.005 for drug effect; Fig. 10E). Thus naloxone did not fully block the U-50488-dependent increase in VT.


Figure 10
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 10. Naloxone does not attenuate U-50488-dependent decrease in VT. Naloxone (0.25 mg/kg) was injected at arrow 1 (immediately after baseline data were obtained), and saline or U-50488 (6.2 mg/kg) was injected at arrow 2 (immediately after 1.0-h exposure to naloxone). There were no time-dependent changes in VE (A) or breathing frequency (B; significant drug effect for breathing frequency was due to different baseline values at time 0 point) or breath duration (D). C: naloxone pretreatment did not block effects of U-50488 on VT, since naloxone injections 1.0 h before U-50488 injections resulted in a 20% increase in VT 2.0 h after injection with U-50488. E: naloxone-U-50488 slightly decreased breaths per episode compared with naloxone-saline. {dagger}P < 0.05 for drug effect.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to systematically test the effects of specific opioid receptor agonists on breathing in awake reptiles. The main findings were that MOR and DOR activation decreased VE by decreasing breathing frequency and breaths per episode, while KOR activation primarily increased VT with variable effects on VE and breathing frequency. Many, but not all, of the agonist effects on breathing were abolished or attenuated, suggesting that drug effects were primarily due to opioid receptor activation.

Complex Changes in Breathing Following Opioid Receptor Activation in Vertebrates

Although opioid drugs are generally believed to decrease respiratory frequency, VT, and chemoreceptor drive, as well as increase upper airway resistance and alter pulmonary mechanics, the effects of opioid agonists on breathing are difficult to predict, because the response depends on the drug (receptor subtype specificity, ability to cross the blood-brain barrier, and pharmacodynamics), route of drug administration (intravenous, intracerebroventricular, intrathecal, and tissue microinjection), state of the animal (conscious vs. anesthetized), and species (type of mammal, reptile, or amphibian), as previously reviewed (42, 44). In addition, peripheral opioid receptors, the function of which is not well understood, may also be activated by systemic administration of opioid drugs (31, 54). Finally, microinjection of specific opioid agonists into the VRG within the brain stem produces either excitatory or inhibitory effects on respiratory frequency and phrenic amplitude, depending on the location of the drug injection and the time following the injection (25, 26). Thus, to better understand how opioid receptor activation modulates breathing in different vertebrates, it is important to establish baseline responses to straightforward systemic opioid drug administration.

MOR activation. DAMGO, an MOR agonist, produced a severe, dose-dependent depression of VE that was completely blocked by prior administration of naloxone. This result is consistent with findings of other studies showing that MOR agonists administered intracerebroventricularly (17, 36), directly on the ventral medullary surface (17) or directly into the VRG (4, 15, 26), produce respiratory depression. Similarly, iontophoresis of MOR agonists onto respiratory neurons throughout the VRG decreases their activity in adult rats and cats (5, 41). In vitro studies show that MOR activation in the brain stem causes respiratory depression (1, 10, 11, 21, 32, 47, 48), although respiratory stimulation has been reported as well (50). In the present study, DAMGO increased inspiratory (but not expiratory) duration, suggesting that DAMGO activated MOR on rhythm-generating neurons controlling inspiration similar to the MOR-dependent hyperpolarization of inspiratory neurons in the mammalian pre-Bötzinger complex (10). However, we cannot rule out that MOR agonists acted indirectly by altering modulatory influences on respiratory rhythm-generating neurons.

DOR activation. The effects of DOR activation on breathing in vertebrates are difficult to interpret because of the different drugs that were used in the studies (for review, see Refs. 25, 44). Systemic or central administration of DADLE or DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) causes respiratory depression via reduced breathing frequency (4, 12, 15, 36, 37, 43). If a MOR antagonist is given before systemic administration of a DOR agonist, respiratory depression is still observed, suggesting that DORs are involved (53). When a more selective DOR agonist, such as DPDPE, was microinjected into the VRG of anesthetized rats, phrenic nerve amplitude was decreased with little change in respiratory frequency (25). In neonatal rat in vitro slice or brain stem-spinal cord preparations, DPDPE has little or no effect on respiratory burst frequency (11, 48, 49) or on respiratory-related neurons (47). In mammals, central DOR activation appears to cause respiratory depression in adult, but not perinatal, mammals.

In ectothermic vertebrates, DPDPE application to isolated lamprey brain stems produces a 30% decrease in respiratory burst frequency with no change in burst shape or amplitude (33). In the present study on awake adult turtles, DOR activation with DPDPE or DADLE produced significant respiratory depression via decreased breathing frequency and increased breath duration. Since DORs are found in abundance within the turtle central nervous system (55), DOR activation probably depressed breathing by acting on neurons controlling rhythm generation, episode frequency, and episode duration. Alternatively, DOR activation may have also altered modulatory influences on respiratory neurons.

Finally, since MOR and DOR activation decreased VE in turtles by decreasing breathing frequency, these turtles would be expected to become hypercapnic and increase VT as a result of increased chemoreceptor drive (20). However, the lack of a significant increase in VT following injection with MOR and DOR agonists suggests that chemoreceptor drive may also have been attenuated, similar to observations in mammals (for review see Ref. 44). In addition, it is not known whether pulmonary mechanics were altered or metabolism was reduced [i.e., decreased oxygen demand (43)] in turtles following MOR- or DOR-induced respiratory depression.

KOR activation. With respect to KOR activation and breathing, there is a wide range of responses. For example, intravenous U-50488 administration decreased phrenic nerve discharge amplitude and duration and also shortened the duration of inspiration and expiration in anesthetized cats (13). Similarly, injections of KOR agonists into the VRG decreased VT and respiratory rate in anesthetized rats (15). In isolated neonatal rat brain stem-spinal cord preparations, U-50488 decreased respiratory burst frequency and amplitude and hyperpolarized VRG inspiratory neurons (47, 48). In contrast, U-50488 administration (intracerebroventricularly, intravenously, or subcutaneously) has very little or no effect on breathing in conscious rodents and monkeys (6, 16, 28). Similarly, in other mammals, administration of various KOR agonists (intracerebroventricularly, intravenously, or subcutaneously) provides antinociception without altering respiration (3, 7, 8). In isolated lamprey brain stem-spinal cord preparations, KOR activation had no effect on lamprey respiratory motor output (32).

In the present study on awake turtles, KOR activation produced highly variable effects on breathing frequency but increased VT in a time-dependent manner. The wide variation in breathing frequency suggests that KOR activation has mixed excitatory and inhibitory effects on different parts of the turtle respiratory control system (e.g., central rhythm generator, central and peripheral chemosensors, mechanosensors, and modulatory circuits) that may be dependent on drug pharmacodynamics. The increased VT in turtles appears to be novel, inasmuch as there are no other reports of increased VT following KOR activation. Because of the robust time-dependent increase in VT, we hypothesize that KOR activation is acting directly on spinal motoneurons controlling pelvic and pectoral girdle breathing movements. Alternatively, KOR receptor activation may decrease inhibitory synaptic inputs onto respiratory-related premotoneurons (22), which would then increase the excitability of glutamatergic premotoneurons projecting to spinal motoneurons, resulting in augmented excitatory drive to spinal motoneurons. Localized application of KOR agonists onto the turtle brain stem or spinal cord may help resolve this question.

Perspectives and Significance

From an evolutionary perspective, our data are consistent with the hypothesis that MOR- and DOR-dependent respiratory depression is conserved in vertebrates. MOR-dependent respiratory depression is observed in lampreys (33), frogs (51, 52), and birds (46), whereas DOR-dependent respiratory depression is observed in adult rats (4, 15, 36, 37), dogs (12, 43), and lampreys (33). To our knowledge, there are no studies on the effects of DOR agonists on breathing in amphibians. In contrast, KOR agonist effects on breathing are not consistent in vertebrates, suggesting that KOR-dependent modulation of respiration is not conserved. From a clinical perspective, analgesic drug administration to vertebrates during conditions that are considered painful to humans is proposed to be the current standard of care in veterinary medicine (35). Alleviation of postsurgical pain in mammals facilitates recovery and healing, reduces morbidity, and contributes to a more rapid return to normal behavior (14, 24). For reptiles, however, little is known with respect to analgesia (2, 35, 39), even though reptiles are commonly maintained as companion animals and heavily represented in zoological and scientific laboratory collections. Unfortunately, opioid drugs that are recommended for use in reptiles (e.g., butorphanol and morphine) may not be effective analgesics and may cause moderate-to-severe respiratory depression (45). One step toward developing rational therapies for providing analgesia without respiratory depression in reptiles is to understand which opioid receptor subtypes provide analgesia and respiratory depression. In the present study, MOR and DOR activation produced respiratory depression. Future studies are needed to determine which opioid receptor agonists provide analgesia. If MOR activation produces respiratory depression and analgesia, then alternative strategies, such as coadministration of drugs that reverse opioid-induced respiratory depression while still maintaining analgesia, such as 5-HT4{alpha} receptor agonists (28; see conflicting evidence in Ref. 27), D1-dopamine receptor agonists (23), and ampakines (40), may be needed.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Foundation Grant IOB 0517302.


   ACKNOWLEDGMENTS
 
The authors thank Andy Gossens for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Johnson, Dept. of Comparative Biosciences, School of Veterinary Medicine, Univ. of Wisconsin, 2015 Linden Dr., Madison, WI 53706 (e-mail: johnsons{at}svm.vetmed.wisc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ballanyi K, Lalley PM, Hoch B, Richter DW. cAMP-dependent reversal of opioid- and prostaglandin-mediated depression of the isolated respiratory network in newborn rats. J Physiol 504: 127–134, 1997.[Abstract/Free Full Text]
  2. Bennett RA. A review of anesthesia and chemical restraint in reptiles. J Zoo Wildl Med 22: 282–303, 1991.[Web of Science]
  3. Castillo R, Kissin I, Bradley EL. Selective {kappa}-opioid agonist for spinal analgesia without the risk of respiratory depression. Anesth Analg 65: 350–354, 1986.[Abstract/Free Full Text]
  4. Chen Z, Hedner J, Hedner T. Substance P-induced respiratory excitation is blunted by {delta}-receptor specific opioids in the rat medulla oblongata. Acta Physiol Scand 157: 165–173, 1996.[CrossRef][Web of Science][Medline]
  5. Denavit-Saubie M, Champagnat J, Zieglgansberger W. Effects of opiates and methionine-enkephalin on pontine and bulbar respiratory neurons of the cat. Brain Res 155: 55–67, 1978.[CrossRef][Web of Science][Medline]
  6. Dosaka-Akita K, Tortella FC, Holaday JW, Long JB. The {kappa}-opioid agonist U-50488H antagonizes respiratory effects of µ-opioid receptor agonists in conscious rats. J Pharmacol Exp Ther 264: 631–637, 1993.[Abstract/Free Full Text]
  7. Freye E, Hartung E, Schenk GK. Bremazocine: an opiate that induces sedation and analgesia without respiratory depression. Anesth Analg 62: 483–488, 1983.[Abstract/Free Full Text]
  8. Fujibayashi K, Kubota-Wantanabe M, Iizuka Y. Effects of R-84760, a selective {kappa}-opioid receptor agonist, on nociception, locomotion and respiration in rats. Arch Int Pharmacodyn Ther 331: 153–162, 1996.[Web of Science][Medline]
  9. Funk GD, Webb CL, Milsom WK. Non-invasive measurement of respiratory tidal volume in aquatic, air-breathing animals. J Exp Biol 126: 519–523, 1986.[Free Full Text]
  10. Gray PA, Rekling JC, Bocchiaro CM, Feldman JL. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the pre-Bötzinger complex. Science 5444: 1566–1568, 1999.
  11. Greer JJ, Carter JE, Al-Zubaidy Z. Opioid depression of respiration in neonatal rats. J Physiol 485: 845–855, 1995.[Abstract/Free Full Text]
  12. Haddad GG, Schaeffer JI, Chang KJ. Opposite effects of the {delta}- and µ-opioid receptor agonists on ventilation in conscious adult dogs. Brain Res 323: 73–82, 1984.[CrossRef][Web of Science][Medline]
  13. Haji A, Takeda R. Effects of a {kappa}-receptor agonist U-50488 on bulbar respiratory neurons and its antagonistic action against the µ-receptor-induced respiratory depression in decerebrate cats. Jpn J Pharmacol 87: 333–337, 2001.[CrossRef][Medline]
  14. Hardie EM, Hansen BD, Carroll GS. Behavior after ovariohysterectomy in the dog: what's normal? Appl Anim Behav Sci 51: 111–128, 1997.[CrossRef][Web of Science]
  15. Hassen AH, Feuerstein G, Faden AI. Selective cardiorespiratory effects mediated by µ-opioid receptors in the nucleus ambiguus. Neuropharmacology 23: 407–415, 1984.[CrossRef][Web of Science][Medline]
  16. Howell LL, Bergman J, Morse WH. Effects of levorphanol and several {kappa}-selective opioids on respiration and behavior in rhesus monkeys. J Pharmacol Exp Ther 245: 364–372, 1988.[Abstract/Free Full Text]
  17. Hurle MA, Mediavilla A, Florez J. Differential respiratory patterns induced by opioids applied to the ventral medullary and dorsal pontine surfaces of cats. Neuropharmacology 24: 597–606, 1985.[CrossRef][Web of Science][Medline]
  18. Inturrisi CE. Clinical pharmacology of opioids for pain. Clin J Pain 18: S3–S13, 2002.[CrossRef][Web of Science][Medline]
  19. Jackson DC. The effect of temperature on ventilation in the turtle, Pseudemys scripta elegans. Respir Physiol 12: 131–140, 1971.[CrossRef][Web of Science][Medline]
  20. Johnson SM, Creighton RJ. Spinal cord injury-induced changes in breathing are not due to supraspinal plasticity in turtles (Pseudemys scripta). Am J Physiol Regul Integr Comp Physiol 289: R1550–R1561, 2005.[Abstract/Free Full Text]
  21. Johnson SM, Smith JC, Feldman JL. Modulation of respiratory rhythm in vitro: role of Gi/o protein-mediated mechanisms. J Appl Physiol 80: 2120–2133, 1996.[Abstract/Free Full Text]
  22. Lalley PM. µ-Opioid receptor agonist effects on medullary respiratory neurons in the cat: evidence for involvement in certain types of ventilatory disturbances. Am J Physiol Regul Integr Comp Physiol 285: R1287–R1304, 2003.[Abstract/Free Full Text]
  23. Lalley PM. D1-dopamine receptor agonists prevent and reverse opiate depression of breathing but not antinociception in the cat. Am J Physiol Regul Integr Comp Physiol 289: R45–R51, 2005.[Abstract/Free Full Text]
  24. Lang JD. Pain: a prelude. Crit Care Clin 15: 1–15, 1999.[CrossRef][Web of Science][Medline]
  25. Lonergan T, Goodchild AK, Christie MJ, Pilowsky PM. Presynaptic {delta}-opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat. Neuroscience 121: 959–973, 2003.[CrossRef][Web of Science][Medline]
  26. Lonergan T, Goodchild AK, Christie MJ, Pilowsky PM. µ-Opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity. Respir Physiol Neurobiol 138: 165–178, 2003.[CrossRef][Web of Science][Medline]
  27. Lötsch J, Skarke C, Schneider A, Hummel T, Geisslinger G. The 5-hydroxytryptamine 4 receptor agonist mosapride does not antagonize morphine-induced respiratory depression. Clin Pharmacol Ther 78: 278–287, 2005.[CrossRef][Web of Science][Medline]
  28. Manzke T, Guenther U, Ponimaskin EG, Haller M, Dutschmann M, Schwarzacher S, Richter DW. 5-HT4(a) receptors avert opioid-induced breathing depression without loss of analgesia. Science 301: 226–229, 2003.[Abstract/Free Full Text]
  29. Matthes HW, Smadja C, Valverde O, Vonesch JL, Foutz AS, Boudinot E, Denavit-Saubie M, Severini C, Negri L, Roques BP, Maldonado R, Kieffer BL. Activity of the {delta}-opioid receptor is partially reduced, whereas activity of the {kappa}-receptor is maintained in mice lacking the µ-receptor. J Neurosci 18: 7285–7295, 1998.[Abstract/Free Full Text]
  30. Milsom WK, Jones DR. The role of vagal afferent information and hypercapnia in control of the breathing pattern in chelonian. J Exp Biol 87: 53–63, 1980.[Abstract/Free Full Text]
  31. Modalen AO, Quiding H, Frey J, Westman L, Lindahl S. A novel molecule (frakefamide) with peripheral opioid properties: the effects on resting ventilation compared with morphine and placebo. Anesth Analg 100: 713–717, 2005.[Abstract/Free Full Text]
  32. Murakoshi T, Suzue T, Tamai S. A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat. Br J Pharmacol 1: 95–104, 1985.
  33. Mutolo D, Bongianni F, Einum J, Dubuc R, Pantaleo T. Opioid-induced depression in the lamprey respiratory network. Neuroscience 150: 720–729, 2007.[CrossRef][Web of Science][Medline]
  34. Onimaru H, Kumagawa Y, Homma I. Respiration-related rhythmic activity in the rostral medulla of newborn rats. J Neurophysiol 96: 55–61, 2006.[Abstract/Free Full Text]
  35. Paul-Murphy J, Ludders JW, Robertson SA, Gaynor JS, Hellyer PW, Wong PL. The need for a cross-species approach to the study of pain in animals. J Am Vet Med Assoc 224: 692–697, 2004.[CrossRef][Web of Science][Medline]
  36. Pazos A, Florez J. Interaction of naloxone with µ- and {delta}-opioid agonists on the respiration of rats. Eur J Pharmacol 87: 309–314, 1983.[CrossRef][Web of Science][Medline]
  37. Pazos A, Florez J. A comparative study in rats of the respiratory depression and analgesia induced by µ- and {delta}-opioid agonists. Eur J Pharmacol 99: 15–21, 1984.[CrossRef][Web of Science][Medline]
  38. Petti MG, Arndt GA. Respiratory depression, nausea, and vomiting, and their relationship to opioids. Curr Opin Anaesthesiol 6: 689–695, 1993.[CrossRef]
  39. Read MR. Evaluation of the use of anesthesia and analgesia in reptiles. J Am Vet Med Assoc 224: 547–552, 2004.[CrossRef][Web of Science][Medline]
  40. Ren J, Poon BY, Tang Y, Funk GD, Greer JJ. Ampakines alleviate respiratory depression in rats. Am J Respir Crit Care Med 174: 1384–1391, 2006.[Abstract/Free Full Text]
  41. Rondouin G, Boudinot E, Champagnat J, Denavit-Saubie M. Effects of sulfonated Leu-enkephalin applied iontophoretically to cat respiratory neurons. Neuropharmacology 20: 963–967, 1981.[CrossRef][Web of Science][Medline]
  42. Santiago TV, Edelman NH. Opioids and breathing. J Appl Physiol 59: 1675–1685, 1985.[Abstract/Free Full Text]
  43. Schaeffer JI, Haddad GG. Regulation of ventilation and oxygen consumption by {delta}- and µ-opioid receptor agonists. J Appl Physiol 59: 959–968, 1985.[Abstract/Free Full Text]
  44. Shook JE, Watkins WD, Camporesi EM. Differential roles of opioid receptors in respiration, respiratory disease, and opiate-induced respiratory depression. Am Rev Respir Dis 142: 895–909, 1990.[Web of Science][Medline]
  45. Sladky KK, Miletic V, Paul-Murphy J, Kinney M, Dallwig R, Johnson SM. Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles (Trachemys scripta). J Am Vet Med Assoc 230: 1356–1362, 2007.[CrossRef][Web of Science][Medline]
  46. Sufka KJ, Hughes RA, Giordano J. Effects of selective opiate antagonists on morphine-induced hyperalgesia in domestic fowl. Pharmacol Biochem Behav 38: 49–54, 1990.[Medline]
  47. Takeda S, Eriksson LI, Yamamoto Y, Joensen H, Onimaru H, Lindahl SGE. Opioid action on respiratory neuron activity of the isolated respiratory network in newborn rats. Anesthesiology 95: 740–749, 2001.[Web of Science][Medline]
  48. Takita K, Herlenius EA, Lindahl SG, Yamamoto Y. Actions of opioids on respiratory activity via activation of brainstem µ-, {delta}-, and {kappa}-receptors: an in vitro study. Brain Res 778: 233–241, 1997.[CrossRef][Web of Science][Medline]
  49. Takita K, Herlenius E, Lindahl SGE, Yamamoto Y. Age- and temperature-dependent effects of opioids on the medulla oblongata respiratory activity: an in vitro study in newborn rat. Brain Res 800: 308–311, 1998.[CrossRef][Web of Science][Medline]
  50. Tanabe A, Fujii T, Onimaru H. Facilitation of respiratory rhythm by a µ-opioid agonist in newborn rat pons-medulla-spinal cord preparations. Neurosci Lett 375: 19–22, 2005.[CrossRef][Web of Science][Medline]
  51. Vasilakos K, Kimura N, Wilson RJ, Remmers JE. Lung and buccal ventilation in the frog: uncoupling coupled oscillators. Physiol Biochem Zool 79: 1010–1018, 2006.[CrossRef][Web of Science][Medline]
  52. Vasilakos K, Wilson RJ, Kimura N, Remmers JE. Ancient gill and lung oscillators may generate the respiratory rhythm of frogs and rats. J Neurobiol 62: 369–385, 2005.[CrossRef][Web of Science][Medline]
  53. Ward SJ, Takemori AE. Determination of the relative involvement of µ-opioid receptors in opioid-induced depression of respiratory rate by use of β-funaltrexamine. Eur J Pharmacol 87: 1–6, 1983.[CrossRef][Web of Science][Medline]
  54. Willette RN, Sapru HN. Peripheral versus central cardiorespiratory effects of morphine. Neuropharmacology 21: 1019–1026, 1982.[CrossRef][Web of Science][Medline]
  55. Xia Y, Haddad GG. Major difference in the expression of {delta}- and µ-opioid receptors between turtle and rat brain. J Comp Neurol 436: 202–210, 2001.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/R1599    most recent
00020.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, S. M.
Right arrow Articles by Wiegel, L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, S. M.
Right arrow Articles by Wiegel, L. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.