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Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression
Accepted Manuscript Title: Inhibition of Protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression Authors: Xiaonan Liang, Zheng Yong, Ruibin Su PII: DOI: Reference:
S0304-3940(18)30292-1 https://doi.org/10.1016/j.neulet.2018.04.029 NSL 33552
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
27-1-2018 7-4-2018 16-4-2018
Please cite this article as: Xiaonan Liang, Zheng Yong, Ruibin Su, Inhibition of Protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression
ARTICLE INFO
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Xiaonan Liang, Zheng Yong*, Ruibin Su*
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State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of
Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China
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Corresponding authors:
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Zheng Yong
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Tel.: +86-10-66931621 Fax: +86-10-68211656
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E-mail address: [email protected]
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Ruibin Su Tel.: +86-10-66931601
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Fax: +86-10-68211656
E-mail address: [email protected]
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Highlights
The mechanisms of opioid-induced respiratory depression were investigated in vivo
PKA and GIRK are involved in fentanyl-induced respiratory depression
PKA affects fentanyl-induced respiratory depression in a cAMP-independent manner
AB STRACT Opioid-induced respiratory depression is a major obstacle to improving the clinical management of
moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects
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of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with
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the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying
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potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects
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on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine
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monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings
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suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could
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represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression.
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Keywords: cAMP, protein kinase A, respiratory network, fentanyl, GIRK
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1. Introduction
Opioid analgesics, such as fentanyl, are currently the workhorses of perioperative analgesia [1, 2].
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Respiratory depression represents the most widely recognized and dangerous side-effect of opioid analgesics and has life-threatening consequences [3, 4]. Clinically, naloxone can be used for the treatment of respiratory depression. However, naloxone eliminates both respiratory depression and the analgesic effects of opioids [5, 6]. It is critical that a pharmacological approach to reverse opioid-induced respiratory depression is developed that does not interfere with analgesia or normal
behavior. However, despite this important clinical problem, the key pathways underlying opioid-induced respiratory depression are not well understood [7, 8].
Studies have shown that the signaling pathways mediating the signals from opioid receptors are
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coupled to Gi proteins [9]. Gi protein-controlled intracellular signaling pathways are activated through two general signal transduction mechanisms: (i) directly through the binding of βγ subunits to channel
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proteins, or (ii) indirectly through the activation of α subunits and reduction in cAMP content, which in
turn depresses PKA activity [10]. However, to date, the specific mechanisms implicated in the action of
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fentanyl on respiratory control centers in the brain have remained poorly defined.
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The respiratory system produces an oscillatory pattern to maintain regular respiratory activity [11].
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Many types of neurons and neurotransmitters participate in the generation and regulation of respiration
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[12, 13]. The fundamental drive to respiration is generated in the brainstem and can be modulated by inputs that include conscious input from the cortex, central, and peripheral chemoreceptors [14]. The
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basic rhythm underling the active inspiratory phase of breathing arises from a specific region of the
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ventrolateral medulla, the preBötzinger complex (preBötC) [15, 16]. Fentanyl and other μ-opiate receptor agonists suppress respiratory activity through direct actions on neurons within the preBötC
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[17]. Opioid receptors are also expressed on carotid bodies and mediate both hypoxia and hypercapnia [18]. However, no study has yet compared the influence of different pathways on fentanyl-induced respiratory depression in vivo. The objective of this study was to investigate the mechanisms involved in opioid-induced respiratory depression in vivo. We examined the effects of cAMP, PKA, and GIRK channels on fentanyl-induced respiratory depression in conscious rats using different pharmacological
agents. Our findings may add to the theoretical basis regarding the mechanisms involved in opioid-induced respiratory depression and help to resolve this clinical problem.
2. Materials and methods
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2.1. Animals All experiments were approved by the appropriate Committee on Animal Care and Use. Adult male
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Sprague–Dawley rats (initially weighing 240–260 g) were purchased from the Beijing Animal Center (Beijing, China). All animals were housed in groups of 5 rats per cage with free access to food and
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water. The room was maintained as a climate-controlled environment (25 ± 1°C) with an alternating
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12-h light-dark cycle (lights on 7 a.m., lights off 7 p.m.). Animals were acclimated to the experimental
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conditions and handled for 3–4 days prior to experiments. All experiments were performed during the
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daytime.
2.2. Pharmacological chemicals
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Fentanyl was purchased from the National Institute of Food and Drug Control with a purity greater
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than 99%. H-89 [N-(2-aminoethyl)-5-isoquinoline-sulfonamide], Tertiapin-Q, 8-Br-cAMP, and rolipram were purchased from Sigma (St. Louis, MO, USA). All chemicals were dissolved in distilled
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water, except for rolipram which was dissolved in 3.75% dimethyl sulfoxide (DMSO), 3.75% Tween 80, and 92.5% distilled water.
2.3. Implantation of intracerebroventricular( i.c.v.) guided cannula Rats were weighed and then anesthetized using pentobarbital sodium (50 mg/kg, intraperitoneally
(i.p.). ). Once each rat was deeply anesthetized, the head was shaved and secured in position in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA) using ear bars and an upper incisor bar. A small incision was made and bregma was located as an anatomical reference point. The skull was carefully exposed and a small hole was drilled. A 23-gauge (23G) stainless steel guide cannula (RWD
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Life Science Co., China) was implanted above the left lateral ventricle of the brain at 1.0 mm posterior, 2.0 mm lateral, and 4.0 mm ventral relative to bregma. Stylets were inserted into the guide cannula to
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prevent clogging. The guide cannula was fixed in position with acrylic dental cement and secured using two skull screws. Rats were kept warm and monitored closely. The rats were allowed to recover for 5
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2.4. I.C.V. drug administration and examinations
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days prior to testing.
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Following a 5-day recovery period after surgical implantation of the stainless-steel guide cannula,
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individual rats received single 10 μL i.c.v. bolus doses of one drug or vehicle into the left lateral ventricle of the brain. Injections were made using a 10 μL syringe at a rate of ~10 μL per 2 min.
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Following the completion of experiments, correct guide cannula placement was assessed in individual
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rats by the injection of 10 μL Evans blue dye solution, which was allowed to diffuse for 10 min. Then, the rats were decapitated and their brains were removed. Correct cannula placement was verified by the
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appearance of dye in the lumen of the lateral ventricle. Only data for animals where the dye met this criterium were used in this study.
2.5. Plethysmography recordings Rats were conditioned to placement in whole-body plethysmograph chambers (WBP; Buxco
Electronics, Inc., Wilmington, NC). Each chamber held one rat. A continuous flow of fresh air (2 L/min) through the plethysmograph chamber maintained a constant temperature and humidity. Experiments were conducted at room temperature (23–25°C). Plethysmographic signals were recorded as changes in the pressure difference between the barometric chamber and reference chamber. Signals were detected
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by transducers, which were then amplified and digitized for computer analysis. Breathing variables were recorded using Ponemah Analysis Modules (DSI). System software was used to calculate the
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respiratory rate (f), tidal volume (VT), minute ventilation (VE = f × VT), inspiratory time (TI), and
expiratory time (TE). To measure respiratory functions, each rat was allowed a 1-h acclimatization
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period in the chamber. A baseline plethysmographic recording was performed for 30 min. Following
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the baseline recordings, the rat was removed from the chamber and a pharmacological agent or vehicle
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was administered. The rat was then returned to the chamber and respiratory functions were recorded at
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5, 10, 15, 20, 25, and 30 min after fentanyl administration. The average respiratory frequency,
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2.6. Statistical analysis
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inspiratory time, expiratory time, and minute volume were calculated.
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Data are expressed as mean ± standard error of the mean (SEM). Differences between treatment groups were evaluated using two-way analysis of variance (ANOVA) followed by the Bonferroni–Dunn test.
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Differences with a P value < 0.05 were deemed statistically significant.
3. Results 3.1. Effects of PKA inhibition on fentanyl-induced ventilatory disturbances At 5–7 days after i.c.v. guide cannula implantation, conscious rats were placed individually into WBP
chambers. The basal respiratory parameters were evaluated, and none of the respiratory functions were affected by guide cannula implantation. No significant difference in behavioral manifestations was detected between the experimental groups before drug treatment. Consistent with previous studies [18], fentanyl administration resulted in a dramatic and short-lasting respiratory depression in rats.
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Administration of vehicle alone did not alter fentanyl-induced respiratory depression, whereas H89 (50 μg/site, i.c.v.) significantly alleviated the reduction in respiratory frequency (F2, 108 = 17.94, P < 0.001),
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increase in inspiratory time (F2, 108 = 6.48, P < 0.01), and changes in expiratory time (F2, 108 = 12.30,
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P < 0.001). The effect was evident at 15 min after fentanyl treatment, and lasted to 30 min (Fig. 1).
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3.2. Effects of GIRK inhibition on fentanyl-induced ventilatory disturbance
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Administration of Tertiapin-Q (i.c.v.), a GIRK channel inhibitor, dose-dependently alleviated
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fentanyl-induced respiratory depression. No significant difference in behavioral manifestations was
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observed between experimental groups before drug treatment. Administration of vehicle alone did not alter fentanyl-induced respiratory depression, whereas Tertiapin-Q significantly and dose-dependently
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(0.5, 2 μg/site) alleviated the fentanyl-induced reduction in respiratory frequency (F2, 114 =5.87, P <
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0.05) and increase in inspiratory time (F2,108=4.97, P < 0.05). The effect on breathing frequency was
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evident at 5 min after fentanyl treatment and lasted to 30 min at a dose of 2 μg/site (Fig. 2).
3.3. Effects of rolipram and 8-Br-cAMP on fentanyl-induced ventilatory disturbance Rolipram and 8-Br-cAMP did not affect fentanyl-induced ventilatory disturbance. No significant difference in behavioral manifestations was observed between each experimental group. The administration of fentanyl induced significant respiratory responses both in rats pre-treated with vehicle
alone and in rats pre-treated with 8-Br-cAMP (100 μg/site, i.c.v.). No significant differences were observed in the respiratory parameters between rats treated with vehicle alone or 8-Br-cAMP during the 30 min after drug administration (P > 0.05). Similarly, pre-treatment with rolipram (1.0, 2.0 mg/kg,
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i.v.) did not alter the respiratory depression induced by fentanyl (Fig. 3–4).
4. Discussions
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The mechanism underlying respiratory depression induced by opioids is unclear. Here, we demonstrate that the PKA inhibitor H89 and GIRK inhibitor Tertiapin-Q alleviated fentanyl-induced respiratory
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depression. The PDE4 inhibitor rolipram and cAMP analog 8-Br-cAMP did not alter fentanyl-induced
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respiratory depression. These results suggest that the PKA and GIRK pathways are both involved in
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fentanyl-induced respiratory depression and that PKA exerts its effects in a cAMP-independent manner.
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Various mechanisms and neuronal sites are involved in opioid-induced respiratory depression [8]. Despite many previous studies on opioid-induced respiratory depression, researchers disagree about
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this issue. Some researchers have found that respiratory depression induced by fentanyl administration
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can be reversed by elevation of cAMP levels [19-21]. Previous studies have also shown that the application of Tertiapin-Q reverses the respiratory rate depression induced by [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) in adult anesthetized rats, and intramuscular administration of fentanyl
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produces a mild inhibitory effect in Girk2-/- mice [22]. Recent studies have also demonstrated that opioid-induced analgesia is linked to the Gi signaling pathway, while respiratory depression may result from activation of the β-arrestin pathway [23, 24].
The reason for the contradictory conclusions is that various mechanisms and neuronal sites are involved in opioid-induced respiratory depression [6]. Different experimental conditions can also lead to different results. For instance, there may be differences in the mechanisms underlying respiratory depression between anesthetized and conscious rats [25]. In vitro, medullary slices containing different
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sections show a distinguishable pattern of respiratory-related rhythm, though this differs from the pattern observed in vivo. The absence of peripheral and descending participation in respiratory
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regulation, low temperature (in vitro preparations are often studied at room temperature), and other differences in the experimental environment may also contribute to differences in results [25]. For
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these reasons, it is not surprising that different results will be produced in different experimental
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conditions. Meanwhile ligand-directed signaling has been suggested as a basis for differences in
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responses to different opioid-induced respiratory effects [26].
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Our findings indicate that the up-regulation of cAMP does not affect fentanyl-induced respiratory depression, and that PKA may be involved in this effect in a cAMP-independent manner. As we know,
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the effects of cAMP cannot be solely attributed to PKA. Exchange protein directly activated by cAMP
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(Epac) and cAMP-gated ion channels also play parts in cAMP signaling as downstream effectors [27]. Recently, the contribution of Epac has become more and more appreciated in different cellular
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processes [28] and in some types of cell they play opposing role [29] or separate roles [30]. In neuronal system, Epac seems to activate distinct molecular signals [28]. In our results inhibition of PKA the effector of cAMP can reverse fentanyl induced respiratory depression but up-regulation of cAMP did not have effects on fentanyl-induced respiratory depression. This effects may be because the effect of Epac is opposite of PKA.
In the respiratory network, multiple neuromodulators are involved in rhythm generation and activity patterning, and they may act simultaneously with other mediators, such as serotonin (5-HT), norepinephrine, adenosine, histamine, substance P, or opioids [12, 31]. Many of these neuromodulators
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act through G protein-regulated intracellular signaling pathways, and the cAMP-PKA system plays an important role in regulating excitability in many types of neurons [32]. A previous study showed that
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inhibition of PKA activity in the preBöC increased phrenic burst frequency [32].Activation of PKA
induces the phosphorylation of various regulatory proteins [33]. In our studies, i.c.v. administration of
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the PKA inhibitor H89 alleviated fentanyl-induced respiratory depression, and this effect may occur
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through inhibition of inhibitory interneurons. This results in respiratory network disinhibition, which
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possibly reverses fentanyl-induced ventilatory depression like activation of 5-HT1A receptors [11]. Our
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results suggest that inhibition of an appropriate PKA signal may reverse opioid-induced respiratory
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depression by altering of intracellular PKA activity via convergent signaling pathways in neurons.
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GIRK channels mediate opioid-induced inhibition of many neural circuits [34]. A previous study
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demonstrated that the application of Tertiapin-Q reverses the respiratory rate depression induced by DAMGO in adult anesthetized rats [22]. In our study, similar changes in respiratory function following
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Tertiapin-Q administration were observed in fentanyl-induced respiratory depression. Together, our results indicate that PKA and GIRK are involved in fentanyl-induced respiratory depression and that PKA can exert this effect in a cAMP-independent manner. The exact mechanism involved in PKA-medicated inhibition of opioid-induced respiratory depression merits further study. Our results add a theoretical basis to the mechanism of opioid-induced respiratory depression and may provide
new ideas for the resolution of this clinical problem. Further research will be necessary to shed more light on the mechanisms of opioid-induced respiratory depression.
Conflict of interest
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The authors have no conflict of interest to declare.
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Acknowledgments
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This study was supported by the Technology Major Project of China (2015ZX09501003).
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Figure legends Fig. 1. Effects of H89 on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are expressed as means ± standard error of the mean (SEM). Arrows indicate the injection of fentanyl. *P < 0.05, **P < 0.01, ***P
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< 0.001, compared with vehicle + fentanyl-treated rats in repeated measures two-way ANOVA with
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Bonferroni post-hoc tests (n =7).
Fig. 2. Effects of the GIRK inhibitor on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are expressed as means ±SEM. Arrows indicate the injection of fentanyl. *P < 0.05, **P < 0.01, ***P < 0.001, compared with vehicle + fentanyl-treated rats in repeated measures two-way ANOVA with Bonferroni post-hoc
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tests (n = 6–9).
Fig. 3. Effects of the PDE4 inhibitor rolipram on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are
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expressed as means ± SEM. Arrows indicate the injection of fentanyl (n = 6–7).
Fig. 4. Effects of the cAMP analog 8-Br-cAMP on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are
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expressed as means ±SEM. Arrows indicate the injection of fentanyl (n = 6–8).
S0304-3940(18)30292-1 https://doi.org/10.1016/j.neulet.2018.04.029 NSL 33552
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
27-1-2018 7-4-2018 16-4-2018
Please cite this article as: Xiaonan Liang, Zheng Yong, Ruibin Su, Inhibition of Protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression
ARTICLE INFO
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Xiaonan Liang, Zheng Yong*, Ruibin Su*
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State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of
Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China
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Corresponding authors:
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Zheng Yong
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Tel.: +86-10-66931621 Fax: +86-10-68211656
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E-mail address: [email protected]
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Ruibin Su Tel.: +86-10-66931601
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Fax: +86-10-68211656
E-mail address: [email protected]
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Highlights
The mechanisms of opioid-induced respiratory depression were investigated in vivo
PKA and GIRK are involved in fentanyl-induced respiratory depression
PKA affects fentanyl-induced respiratory depression in a cAMP-independent manner
AB STRACT Opioid-induced respiratory depression is a major obstacle to improving the clinical management of
moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects
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of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with
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the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying
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potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects
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on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine
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monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings
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suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could
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represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression.
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Keywords: cAMP, protein kinase A, respiratory network, fentanyl, GIRK
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1. Introduction
Opioid analgesics, such as fentanyl, are currently the workhorses of perioperative analgesia [1, 2].
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Respiratory depression represents the most widely recognized and dangerous side-effect of opioid analgesics and has life-threatening consequences [3, 4]. Clinically, naloxone can be used for the treatment of respiratory depression. However, naloxone eliminates both respiratory depression and the analgesic effects of opioids [5, 6]. It is critical that a pharmacological approach to reverse opioid-induced respiratory depression is developed that does not interfere with analgesia or normal
behavior. However, despite this important clinical problem, the key pathways underlying opioid-induced respiratory depression are not well understood [7, 8].
Studies have shown that the signaling pathways mediating the signals from opioid receptors are
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coupled to Gi proteins [9]. Gi protein-controlled intracellular signaling pathways are activated through two general signal transduction mechanisms: (i) directly through the binding of βγ subunits to channel
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proteins, or (ii) indirectly through the activation of α subunits and reduction in cAMP content, which in
turn depresses PKA activity [10]. However, to date, the specific mechanisms implicated in the action of
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fentanyl on respiratory control centers in the brain have remained poorly defined.
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The respiratory system produces an oscillatory pattern to maintain regular respiratory activity [11].
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Many types of neurons and neurotransmitters participate in the generation and regulation of respiration
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[12, 13]. The fundamental drive to respiration is generated in the brainstem and can be modulated by inputs that include conscious input from the cortex, central, and peripheral chemoreceptors [14]. The
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basic rhythm underling the active inspiratory phase of breathing arises from a specific region of the
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ventrolateral medulla, the preBötzinger complex (preBötC) [15, 16]. Fentanyl and other μ-opiate receptor agonists suppress respiratory activity through direct actions on neurons within the preBötC
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[17]. Opioid receptors are also expressed on carotid bodies and mediate both hypoxia and hypercapnia [18]. However, no study has yet compared the influence of different pathways on fentanyl-induced respiratory depression in vivo. The objective of this study was to investigate the mechanisms involved in opioid-induced respiratory depression in vivo. We examined the effects of cAMP, PKA, and GIRK channels on fentanyl-induced respiratory depression in conscious rats using different pharmacological
agents. Our findings may add to the theoretical basis regarding the mechanisms involved in opioid-induced respiratory depression and help to resolve this clinical problem.
2. Materials and methods
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2.1. Animals All experiments were approved by the appropriate Committee on Animal Care and Use. Adult male
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Sprague–Dawley rats (initially weighing 240–260 g) were purchased from the Beijing Animal Center (Beijing, China). All animals were housed in groups of 5 rats per cage with free access to food and
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water. The room was maintained as a climate-controlled environment (25 ± 1°C) with an alternating
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12-h light-dark cycle (lights on 7 a.m., lights off 7 p.m.). Animals were acclimated to the experimental
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conditions and handled for 3–4 days prior to experiments. All experiments were performed during the
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daytime.
2.2. Pharmacological chemicals
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Fentanyl was purchased from the National Institute of Food and Drug Control with a purity greater
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than 99%. H-89 [N-(2-aminoethyl)-5-isoquinoline-sulfonamide], Tertiapin-Q, 8-Br-cAMP, and rolipram were purchased from Sigma (St. Louis, MO, USA). All chemicals were dissolved in distilled
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water, except for rolipram which was dissolved in 3.75% dimethyl sulfoxide (DMSO), 3.75% Tween 80, and 92.5% distilled water.
2.3. Implantation of intracerebroventricular( i.c.v.) guided cannula Rats were weighed and then anesthetized using pentobarbital sodium (50 mg/kg, intraperitoneally
(i.p.). ). Once each rat was deeply anesthetized, the head was shaved and secured in position in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA) using ear bars and an upper incisor bar. A small incision was made and bregma was located as an anatomical reference point. The skull was carefully exposed and a small hole was drilled. A 23-gauge (23G) stainless steel guide cannula (RWD
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Life Science Co., China) was implanted above the left lateral ventricle of the brain at 1.0 mm posterior, 2.0 mm lateral, and 4.0 mm ventral relative to bregma. Stylets were inserted into the guide cannula to
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prevent clogging. The guide cannula was fixed in position with acrylic dental cement and secured using two skull screws. Rats were kept warm and monitored closely. The rats were allowed to recover for 5
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2.4. I.C.V. drug administration and examinations
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days prior to testing.
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Following a 5-day recovery period after surgical implantation of the stainless-steel guide cannula,
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individual rats received single 10 μL i.c.v. bolus doses of one drug or vehicle into the left lateral ventricle of the brain. Injections were made using a 10 μL syringe at a rate of ~10 μL per 2 min.
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Following the completion of experiments, correct guide cannula placement was assessed in individual
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rats by the injection of 10 μL Evans blue dye solution, which was allowed to diffuse for 10 min. Then, the rats were decapitated and their brains were removed. Correct cannula placement was verified by the
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appearance of dye in the lumen of the lateral ventricle. Only data for animals where the dye met this criterium were used in this study.
2.5. Plethysmography recordings Rats were conditioned to placement in whole-body plethysmograph chambers (WBP; Buxco
Electronics, Inc., Wilmington, NC). Each chamber held one rat. A continuous flow of fresh air (2 L/min) through the plethysmograph chamber maintained a constant temperature and humidity. Experiments were conducted at room temperature (23–25°C). Plethysmographic signals were recorded as changes in the pressure difference between the barometric chamber and reference chamber. Signals were detected
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by transducers, which were then amplified and digitized for computer analysis. Breathing variables were recorded using Ponemah Analysis Modules (DSI). System software was used to calculate the
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respiratory rate (f), tidal volume (VT), minute ventilation (VE = f × VT), inspiratory time (TI), and
expiratory time (TE). To measure respiratory functions, each rat was allowed a 1-h acclimatization
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period in the chamber. A baseline plethysmographic recording was performed for 30 min. Following
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the baseline recordings, the rat was removed from the chamber and a pharmacological agent or vehicle
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was administered. The rat was then returned to the chamber and respiratory functions were recorded at
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5, 10, 15, 20, 25, and 30 min after fentanyl administration. The average respiratory frequency,
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2.6. Statistical analysis
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inspiratory time, expiratory time, and minute volume were calculated.
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Data are expressed as mean ± standard error of the mean (SEM). Differences between treatment groups were evaluated using two-way analysis of variance (ANOVA) followed by the Bonferroni–Dunn test.
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Differences with a P value < 0.05 were deemed statistically significant.
3. Results 3.1. Effects of PKA inhibition on fentanyl-induced ventilatory disturbances At 5–7 days after i.c.v. guide cannula implantation, conscious rats were placed individually into WBP
chambers. The basal respiratory parameters were evaluated, and none of the respiratory functions were affected by guide cannula implantation. No significant difference in behavioral manifestations was detected between the experimental groups before drug treatment. Consistent with previous studies [18], fentanyl administration resulted in a dramatic and short-lasting respiratory depression in rats.
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Administration of vehicle alone did not alter fentanyl-induced respiratory depression, whereas H89 (50 μg/site, i.c.v.) significantly alleviated the reduction in respiratory frequency (F2, 108 = 17.94, P < 0.001),
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increase in inspiratory time (F2, 108 = 6.48, P < 0.01), and changes in expiratory time (F2, 108 = 12.30,
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P < 0.001). The effect was evident at 15 min after fentanyl treatment, and lasted to 30 min (Fig. 1).
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3.2. Effects of GIRK inhibition on fentanyl-induced ventilatory disturbance
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Administration of Tertiapin-Q (i.c.v.), a GIRK channel inhibitor, dose-dependently alleviated
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fentanyl-induced respiratory depression. No significant difference in behavioral manifestations was
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observed between experimental groups before drug treatment. Administration of vehicle alone did not alter fentanyl-induced respiratory depression, whereas Tertiapin-Q significantly and dose-dependently
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(0.5, 2 μg/site) alleviated the fentanyl-induced reduction in respiratory frequency (F2, 114 =5.87, P <
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0.05) and increase in inspiratory time (F2,108=4.97, P < 0.05). The effect on breathing frequency was
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evident at 5 min after fentanyl treatment and lasted to 30 min at a dose of 2 μg/site (Fig. 2).
3.3. Effects of rolipram and 8-Br-cAMP on fentanyl-induced ventilatory disturbance Rolipram and 8-Br-cAMP did not affect fentanyl-induced ventilatory disturbance. No significant difference in behavioral manifestations was observed between each experimental group. The administration of fentanyl induced significant respiratory responses both in rats pre-treated with vehicle
alone and in rats pre-treated with 8-Br-cAMP (100 μg/site, i.c.v.). No significant differences were observed in the respiratory parameters between rats treated with vehicle alone or 8-Br-cAMP during the 30 min after drug administration (P > 0.05). Similarly, pre-treatment with rolipram (1.0, 2.0 mg/kg,
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i.v.) did not alter the respiratory depression induced by fentanyl (Fig. 3–4).
4. Discussions
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The mechanism underlying respiratory depression induced by opioids is unclear. Here, we demonstrate that the PKA inhibitor H89 and GIRK inhibitor Tertiapin-Q alleviated fentanyl-induced respiratory
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depression. The PDE4 inhibitor rolipram and cAMP analog 8-Br-cAMP did not alter fentanyl-induced
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respiratory depression. These results suggest that the PKA and GIRK pathways are both involved in
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fentanyl-induced respiratory depression and that PKA exerts its effects in a cAMP-independent manner.
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Various mechanisms and neuronal sites are involved in opioid-induced respiratory depression [8]. Despite many previous studies on opioid-induced respiratory depression, researchers disagree about
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this issue. Some researchers have found that respiratory depression induced by fentanyl administration
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can be reversed by elevation of cAMP levels [19-21]. Previous studies have also shown that the application of Tertiapin-Q reverses the respiratory rate depression induced by [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) in adult anesthetized rats, and intramuscular administration of fentanyl
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produces a mild inhibitory effect in Girk2-/- mice [22]. Recent studies have also demonstrated that opioid-induced analgesia is linked to the Gi signaling pathway, while respiratory depression may result from activation of the β-arrestin pathway [23, 24].
The reason for the contradictory conclusions is that various mechanisms and neuronal sites are involved in opioid-induced respiratory depression [6]. Different experimental conditions can also lead to different results. For instance, there may be differences in the mechanisms underlying respiratory depression between anesthetized and conscious rats [25]. In vitro, medullary slices containing different
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sections show a distinguishable pattern of respiratory-related rhythm, though this differs from the pattern observed in vivo. The absence of peripheral and descending participation in respiratory
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regulation, low temperature (in vitro preparations are often studied at room temperature), and other differences in the experimental environment may also contribute to differences in results [25]. For
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these reasons, it is not surprising that different results will be produced in different experimental
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conditions. Meanwhile ligand-directed signaling has been suggested as a basis for differences in
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responses to different opioid-induced respiratory effects [26].
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Our findings indicate that the up-regulation of cAMP does not affect fentanyl-induced respiratory depression, and that PKA may be involved in this effect in a cAMP-independent manner. As we know,
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the effects of cAMP cannot be solely attributed to PKA. Exchange protein directly activated by cAMP
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(Epac) and cAMP-gated ion channels also play parts in cAMP signaling as downstream effectors [27]. Recently, the contribution of Epac has become more and more appreciated in different cellular
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processes [28] and in some types of cell they play opposing role [29] or separate roles [30]. In neuronal system, Epac seems to activate distinct molecular signals [28]. In our results inhibition of PKA the effector of cAMP can reverse fentanyl induced respiratory depression but up-regulation of cAMP did not have effects on fentanyl-induced respiratory depression. This effects may be because the effect of Epac is opposite of PKA.
In the respiratory network, multiple neuromodulators are involved in rhythm generation and activity patterning, and they may act simultaneously with other mediators, such as serotonin (5-HT), norepinephrine, adenosine, histamine, substance P, or opioids [12, 31]. Many of these neuromodulators
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act through G protein-regulated intracellular signaling pathways, and the cAMP-PKA system plays an important role in regulating excitability in many types of neurons [32]. A previous study showed that
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inhibition of PKA activity in the preBöC increased phrenic burst frequency [32].Activation of PKA
induces the phosphorylation of various regulatory proteins [33]. In our studies, i.c.v. administration of
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the PKA inhibitor H89 alleviated fentanyl-induced respiratory depression, and this effect may occur
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through inhibition of inhibitory interneurons. This results in respiratory network disinhibition, which
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possibly reverses fentanyl-induced ventilatory depression like activation of 5-HT1A receptors [11]. Our
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results suggest that inhibition of an appropriate PKA signal may reverse opioid-induced respiratory
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depression by altering of intracellular PKA activity via convergent signaling pathways in neurons.
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GIRK channels mediate opioid-induced inhibition of many neural circuits [34]. A previous study
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demonstrated that the application of Tertiapin-Q reverses the respiratory rate depression induced by DAMGO in adult anesthetized rats [22]. In our study, similar changes in respiratory function following
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Tertiapin-Q administration were observed in fentanyl-induced respiratory depression. Together, our results indicate that PKA and GIRK are involved in fentanyl-induced respiratory depression and that PKA can exert this effect in a cAMP-independent manner. The exact mechanism involved in PKA-medicated inhibition of opioid-induced respiratory depression merits further study. Our results add a theoretical basis to the mechanism of opioid-induced respiratory depression and may provide
new ideas for the resolution of this clinical problem. Further research will be necessary to shed more light on the mechanisms of opioid-induced respiratory depression.
Conflict of interest
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The authors have no conflict of interest to declare.
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Acknowledgments
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This study was supported by the Technology Major Project of China (2015ZX09501003).
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Figure legends Fig. 1. Effects of H89 on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are expressed as means ± standard error of the mean (SEM). Arrows indicate the injection of fentanyl. *P < 0.05, **P < 0.01, ***P
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< 0.001, compared with vehicle + fentanyl-treated rats in repeated measures two-way ANOVA with
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Bonferroni post-hoc tests (n =7).
Fig. 2. Effects of the GIRK inhibitor on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are expressed as means ±SEM. Arrows indicate the injection of fentanyl. *P < 0.05, **P < 0.01, ***P < 0.001, compared with vehicle + fentanyl-treated rats in repeated measures two-way ANOVA with Bonferroni post-hoc
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tests (n = 6–9).
Fig. 3. Effects of the PDE4 inhibitor rolipram on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are
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expressed as means ± SEM. Arrows indicate the injection of fentanyl (n = 6–7).
Fig. 4. Effects of the cAMP analog 8-Br-cAMP on 60 μg/kg fentanyl-induced respiratory depression. Respiratory frequency, inspiratory time, expiratory time, and minute volume in conscious rats are
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expressed as means ±SEM. Arrows indicate the injection of fentanyl (n = 6–8).