L-cysteine ethylester reverses the adverse effects of morphine on breathing and arterial blood-gas chemistry while minimally affecting antinociception in unanesthetized rats

Abstract

L-cysteine ethylester (L-CYSee) is a membrane-permeable analogue of L-cysteine with a variety of pharmacological effects. The purpose of this study was to determine the effects of L-CYSee on morphine-induced changes in ventilation, arterial-blood gas (ABG) chemistry, Alveolar-arterial (A-a) gradient (i.e., a measure of the index of alveolar gas-exchange), antinociception and sedation in male Sprague Dawley rats. An injection of morphine (10 mg/kg, IV) produced adverse effects on breathing, including sustained decreases in minute ventilation. L-CYSee (500 μmol/kg, IV) given 15 min later immediately reversed the actions of morphine. Another injection of L-CYSee (500 μmol/kg, IV) after 15 min elicited more pronounced excitatory ventilatory responses. L-CYSee (250 or 500 μmol/kg, IV) elicited a rapid and prolonged reversal of the actions of morphine (10 mg/kg, IV) on ABG chemistry (pH, pCO₂, pO₂, sO₂) and A-a gradient. L-serine ethylester (an oxygen atom replaces the sulfur; 500 μmol/kg, IV), was ineffective in all studies. L-CYSee (500 μmol/kg, IV) did not alter morphine (10 mg/kg, IV)-induced sedation, but slightly reduced the overall duration of morphine (5 or 10 mg/kg, IV)-induced analgesia. In summary, L-CYSee rapidly overcame the effects of morphine on breathing and alveolar gas-exchange, while not affecting morphine sedation or early-stage analgesia. The mechanisms by which L-CYSee modulates morphine depression of breathing are unknown, but appear to require thiol-dependent processes.

1. Introduction

The clinical efficacies of opioid analgesics, such as morphine, are compromised of adverse effects on breathing and gas-exchange in alveoli [1-9]. Opioid-induced respiratory depression (OIRD) can be partially overcome by opioid receptor antagonists, such as naloxone (Narcan), but they also reverse opioid-induced analgesia, which is problematic when maintaining analgesia is essential in scenarios of severe injury and pre/post-surgery [1-5]. Several classes of therapeutics, without opioid receptor antagonist actions, have been tested against OIRD (Supplemental Table 1 in reference 9 for citations) [7,8]. Most of these drugs were not tested in clinical trials or failed to progress satisfactorily in such trials due to poor efficacy or severity of side-effects, although the NMDA receptor antagonist, esketamine, and several ampakines are being currently evaluated [3,4,6-9]. At present, the mechanisms and/or sites of action that are needed for development of efficacious agents that reverse OIRD while preserving analgesia are unknown [3,6,7].

Morphine diminishes entry of L-cysteine into neurons by blockade of the type 3 excitatory amino acid transporter, which plays an essential role in the cellular uptake of this amino acid [10,11]. Trivedi et al. [11] proposed that the subsequent changes in the redox environment of the neurons to a more oxidative status without intracellular L-cysteine, and/or loss of involvement of L-cysteine in a host of signaling-metabolic pathways [12-21], drives the development of morphine addiction. Hypothesizing that opioid-induced inhibition of L-cysteine entry into cells may be involved in OIRD, it is possible cell-permeable methylester or ethylester derivatives of cysteine-containing compounds, which deliver thiolesters into cells, may be a therapeutic approach to preventing and/or reversing OIRD. As shown in Supplemental Table 21 of Getsy et al. [9], there are monothiol (reduced) and disulfide (oxidized) L-thiolesters that readily enter neurons and other cell types in the central nervous system. The mechanism of action of L-thiolesters may involve (1) interaction of L-thiolesters with proteins in plasma, on plasma membranes, and/or inside of cells, (2) generation of mixed disulfides [22-24] and thiol adducts, such as L-cysteine:D-glucose in the blood [25-27], (3) generation of S-thiolated proteins in cells [28-33], (4) altered redox status (e.g., switching of cysteine-cystine status), and functionality of proteins in plasma membrane, including voltage-gated (Kv_1.2) K⁺-channels [34], (5) redox modulation of intracellular proteins [35-40], (6) conversion of L-thiolesters to parent L-thiols by carboxylesterases [41-44] causing elevations in intracellular levels of L-thiols, which become available to enter metabolic pathways, including those generating hydrogen sulfide via L-cysteine aminotransferase and cystathionine γ-lyase in tissues [45-47], including carotid bodies [48], (7) enzymatic (cysteine dioxygenase) production of cysteine-sulfenic acid, cysteine-sulfinic acid and cysteine-sulfonic acid [49-52], and (8) formation of S-nitroso-L-cysteine, an endogenous S-nitrosothiol [53-55] with key roles in intracellular signaling pathways [56-60], including those in control of cardiovascular and ventilatory processes [60-67], and those modulating OIRD [68,69].

As shown in Supplemental Table 1, we have determined efficacies of N-acetyl-L-cysteine (L-NAC) [70], several L- and D-thiolesters [71-77], the antioxidant/superoxide anion scavenger, Tempol [78,79], and the nitric oxide synthase inhibitor, N^G-nitro-L-arginine methylester (L-NAME) [67], in modulating OIRD elicited by morphine or fentanyl in freely-moving or anesthetized male Sprague Dawley rats to begin understanding the structure-activity relationships with respect to OIRD, and potential influence of these compounds on anesthesia. Note, at the present time, none of the thiols or Tempol described in Supplemental Table 1, adversely affected opioid-induced analgesia [68-79]. The major findings from these studies included that (1) bolus injections of L-NAC elicited a sustained reversal of OIRD, and depression of arterial blood-gas (ABG) chemistry (pH, pCO₂, pO₂, sO₂) and Alveolar-arterial (A-a) gradient (i.e., a measure of the index of alveolar gas-exchange) in unanesthetized rats receiving continuous fentanyl infusion [70], (2) L-cysteine ethylester (L-CYSee) reversed the effects of morphine on ABG chemistry in anesthetized tracheotomized rats [71], (3) ventilatory depression caused by injection of morphine in unanesthetized rats was blunted when receiving continuous infusion of L-CYSee [72], (4) injections of L-cysteine methylester (L-CYSme), D-cysteine ethylester (D-CYSee), D-cystine diethylester (L-CYSdiee), or D-cystine dimethylester (D-CYSdime) reversed the effects of morphine on breathing and ABG chemistry in unanesthetized rats [73-76], and (5) the adverse actions of fentanyl on breathing and ABG chemistry are diminished in conscious rats that received a prior injection of L-glutathione ethylester (L-GSHee) [77]. Data from studies using L- or D-thiolester compounds, show that the parent thiols, such as L-cysteine, D-cysteine, D-cystine and L-glutathione, were inactive [71-75], which suggests that the abilities of L-, D-thiolesters to prevent/reverse OIRD involves entry into neurons and other (e.g., skeletal muscle) cells to initiate intracellular signaling events to prevent/reverse OIRD. Unlike opioid receptor antagonists, it appears that the L- and D-thiolesters reverse the actions of opioids on breathing and ABG chemistry by mechanisms other than direct/allosteric block of opioid receptors [73-77]. In addition, as seen in Supplemental Table 1, studies designed to build on the L-, D-thiolester findings found that (1) the cardiorespiratory depressant effects of fentanyl were attenuated in anesthetized rats that had received prior injection of a free radical/superoxide anion scavenger, Tempol [78], (2) Tempol reversed the negative effects of morphine on ABG chemistry and tissue O₂ saturation in rats [79], (3) nitrosyl factors play a vital role in the ventilatory depressant effects of fentanyl in freely-moving rats [67], (4) the adverse effects of fentanyl on breathing were blunted in freely-moving rats receiving infusion of S-nitroso-L-cysteine [68], and (5) the adverse effects of morphine on breathing and ABG chemistry were blunted in anesthetized rats receiving infusion of S-nitroso-L-cysteine [69].

The hypothesis of the present study was to discover whether or not L-CYSee will overcome the adverse actions of morphine in freely-moving rats. The first objective was to determine the effects of L-CYSee (500 μmol/kg, IV) on morphine (10 mg/kg, IV)-induced changes on breathing, A-a gradient, ABG chemistry, analgesia, and sedation. The second objective was to determine whether L-CYSee overcomes the adverse effects of morphine on the ventilatory responses to a subsequent hypoxic-hypercapnic (HH) gas challenge, and those ventilatory responses upon return to room-air. The doses of morphine and L-CYSee are based on previous studies showing the profound effects of this particular dose of morphine on breathing, and the efficacy of 500 μmol/kg doses of L,D-thiolesters [73-77].

2. Methods

2.1. Permissions, rats, and surgical procedures

All studies were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals (NIH Publication No. 80–23) revised in 2011, and in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org/). All protocols involving rats were approved by the Animal Care and Use Committees of Galleon Pharmaceuticals, Case Western Reserve University, and the University of Virginia. Adult male Sprague Dawley rats were purchased from Harlan Industries (Madison, WI, USA). After five days of recovery from transportation, groups of rats were anesthetized under 2–3% isoflurane and implanted with a jugular vein catheter only, or both a jugular vein catheter and a femoral artery catheter, as detailed previously [80-82]. The rats were given four days to recover from surgery before use. All femoral arterial catheters were flushed daily with a heparin solution (50 units heparin in 0.1 M, pH 7.4 phosphate-buffered saline). All arterial and venous catheters were flushed with 0.3 ml of phosphate-buffered saline (0.1 M, pH 7.4) 3–4 h before commencement of the study. The pH of all stock solutions of vehicle, L-CYSee, L-cysteine, L-SERee and L-serine were adjusted to pH of 7.2 with 0.25 M NaOH. All studies were done in a quiet room with relative humidity of 50 ± 2%, and room temperature of 21.3 ± 0.2 °C. The antinociception and ABG chemistry studies were done in separate groups of rats to not compromise the ventilatory recordings. Plethysmography recordings, antinociception recording sessions, and arterial blood sampling studies (ABG assays) were performed by an investigator who injected the opioid, vehicle, or test drugs. The syringes with vehicle or test drug were made up by another investigator, such that the investigator running the study was blind to the treatment protocol. Injectable (liquid) form of (+)-morphine sulfate (10 mg/ml) was from Baxter Healthcare Corporation (Deerfield, IL, USA). L-CYSee HCl powder (product number: C121908; PubChem Substance ID: 24892386), L-cysteine HCl monohydrate powder (product number: 30129; PubChem Substance ID: 57648412), L-SERee HCl powder (product number: 223123; PubChem Substance ID: 24853367), and L-serine HCl powder (product number: S4500; PubChem Substance ID: 24899605) were from Sigma-Aldrich (St. Louis, MO, USA), and divided into 100 mg amounts under N₂ gas and stored at 4 °C. Solutions of L-CYSee (dissolved in saline and brought to pH 7.2 with 0.1 M NaOH at room temperature) were prepared immediately before injection. In every case, the data files resulting from each study were collated and analyzed by another investigator. Note, that each rat used here received only one treatment and was not re-used in any study. Figures and diagrams describing the plethysmography set-up can be found at the supplier’s (Data Sciences International) site at https://www.datasci.com/products/buxco-respiratory-products, and figures and diagrams for the analgesia testing equipment can be found at the supplier’s (IITC Life Science Inc., USA) site at http://www.iitcinc.com/Analgesia.html. As seen in Supplemental Table 2, 31 groups of rats (n = 211 rats in total) were used in this study. We first analyzed the effects of L-CYSee, a cell-permeant thiol (sulfur atom-containing ester), in plethysmography studies. To test the importance of the sulfur atom in the thiol ester, we determined the effects of L-SERee, which is a cell-penetrant oxygen-containing rather than sulfur-containing ester. On the basis of the findings showing the efficacy of L-CYSee, but not L-SERee, we wanted to determine whether or not the ability of L-CYSee to reverse the adverse effects of morphine on ABG chemistry was due to enhanced cell-penetration, we futher examined the effects of L-cysteine. A full panel of studies using L-CYSee, L-cysteine, L-SERee and L-serine was performed in tail-flick latency (TFL) studies to better characterize the effects of L-CYSee on morphine analgesia. On the basis of the TFL findings, we thought it necessary to only study the effects of L-CYSee and L-cysteine in hind-paw withdrawal latency (HPL) studies. Finally, a full panel of studies using L-CYSee, L-cysteine, L-SERee and L-serine was performed in righting reflex studies to better characterize the effects of L-CYSee on morphine sedation.

2.2. Protocols for whole body plethysmography

Ventilatory parameters were recorded continuously in unrestrained freely-moving rats using a whole body plethysmography system (PLY3223; Data Sciences International, St. Paul, MN) as detailed previously [67-82]. The directly recorded, and calculated (derived) parameters are defined in Supplemental Table 3. The ventilatory parameters and abbreviations are: frequency of breathing (Freq), tidal volume (TV), minute ventilation (MV), inspiratory time (Ti), expiratory time (Te), Ti/Te, end inspiratory pause (EIP), end expiratory pause (EEP), peak inspiratory flow (PIF), peak expiratory flow (PEF), PIF/PEF, expiratory flow at 50% expired TV (EF₅₀), relaxation time (RT), inspiratory drive (TV/Ti), expiratory drive (TV/Te), apneic pause [(Te/RT)− 1], inspiratory delay (Te-RT), non-eupneic breathing index (NEBI), and NEBI corrected for Freq (NEBI/Freq). A diagram adapted from Lomask [83] shows the relationship between some directly recorded parameters Supplemental Fig. 1. On the day of the study, each rat was placed in a plethysmography chamber and allowed 60 min to acclimatize before resting (i.e., baseline, pre) ventilatory parameter values were defined. While rats were acclimatizing to the chambers, they were placed in one of two studies. Study 1: Two groups of rats received an injection of morphine (10 mg/kg, IV) and after 15 min, one group (82.9 ± 0.4 days of age; 337 ± 3 g body weight; n = 6) received an injection of vehicle (saline), and the other group (83.9 ± 0.6 days of age; 338 ± 3 g body weight; n = 6) an injection of L-CYSee (500 μmol/kg, IV). The rats then received a second injection of vehicle or L-CYSee (500 μmol/kg, IV) 15 min later. Ventilatory parameters were monitored for 60 min after the second injections of vehicle or L-CYSee (500 μmol/kg, IV). Sixty min after the second injection, the rats were given a hypoxic-hypercapnic rebreathing (HH) challenge [84] in which airflow through each chamber was turned off for 65 min allowing the rats to rebreathe air that gradually became more hypoxic and more hypercapnic over time [84]. Upon return to room-air, ventilatory parameters were recorded for a further 25 min. Study 2: Two groups of rats received a bolus injection of morphine (10 mg/kg, IV) and after 15 min, one group (83.4 ± 0.5 days of age; 336 ± 3 g body weight; n = 6) received an injection of vehicle (saline), and the other group (83.1 ± 0.6 days of age; 338 ± 3 g body weight; n = 6) received an injection of L-SERee (500 μmol/kg, IV). The rats then received a second injection of vehicle or L-SERee (500 μmol/kg, IV) 15 min later. Sixty min after the second injection, the rats were exposed to HH challenge for 60 min (this HH challenge is the same challenge as in study 1), and upon return to room-air, parameters were recorded for a further 25 min. Body weights of all groups were similar to one another (p > 0.05 for all comparisons), and thus ventilatory parameters related to volumes (e.g., TV, PIF, PEF, EF₅₀) are given without body weight corrections. The FinePointe (DSI) software constantly corrected digitized values originating from actual waveforms for alterations in chamber temperature and humidity. Pressure changes associated with respiratory waveforms were converted to volumes (e.g., TV, PIF, PEF, EF₅₀) using algorithms of Epstein et al. [84,85]. Factoring in chamber temperature and humidity, cycle analyzers filtered the acquired signals and FinePointe algorithms generated an array of box flow data that identified a waveform segment as an acceptable breath. From that data array, the minimum and maximum box flow values were obtained and multiplied by a compensation factor provided by the selected algorithm [85,86], thus producing TV, PIF, and PEF values used to determine non-eupneic breathing events expressed as non-eupneic breathing index (NEBI, % of non-eupneic breathing events per epoch) [87]. Apneic pause was also calculated as (Expiratory Time/Relaxation Time) − 1 [76].

2.3. Protocols for blood gas measurements and determination of Arterial-alveolar gradient

We recorded changes in ABG chemistry values as detailed previously [68-80]. Briefly, the arterial blood samples for ABG analyses were taken from the femoral arterial catheter. Blood was drawn into a syringe until it was evident that arterial blood untainted by saline was at the tip of the exteriorized catheter. Then a sample of arterial blood (100 μL) was drawn into another syringe for analysis, and the contents of the first syringe were reinjected into the rats followed by a 300 μL of saline to clear the catheter (i.e., flush any blood back into the rat). From the blood samples we gathered measurements included pH, pCO₂, pO₂, sO₂ and A-a gradients elicited by (1) an injection of morphine (10 mg/kg, IV). and (2) after 15 min by an injection of vehicle (saline, IV; 83.1 ± 0.6 days of age; 337 ± 2 g body weight), L-CYSee (500 μmol/kg, IV; 82.5 ± 0.7 days of age; 335 ± 3 g body weight), L-cysteine (500 μmol/kg, IV; 84.0 ± 0.7 days or age; 339 ± 3 g body weight), L-SERee (500 μmol/kg, IV; 82.6 ± 0.5 days of age; 336 ± 3 g body weight), or L-serine (500 μmol/kg, IV; 82.9 ± 0.7 days of age; 338 ± 2 g body weight) in 3 different sets of unanesthetized freely-moving rats (n = 9 rats per group). Samples of arterial blood (100 μL) were taken from the femoral arterial catheter 15 min before and 15 min after injection of morphine (10 mg/kg, IV). Fifteen minutes after morphine injection, the rats then immediately received an injection of vehicle or test agents, and blood samples were taken at 5, 15, 30 and 45 min timepoints. The pH, pCO₂, pO₂ and sO₂ were determined by a Radiometer blood-gas analyzer (ABL800 FLEX). The A-a gradient defines differences between alveolar and arterial blood O₂ concentrations [87-89]. For instance, a fall in PaO₂, without a concomitant alteration in A-a gradient is the result of hypo-ventilation, whereas a decrease in PaO₂ with a concomitant increase in A-a gradient, indicates on-going mismatch in ventilation-perfusion in alveoli [87-89]. A-a gradient = PAO₂ − PaO₂, where PAO₂ is the partial pressure (p) of alveolar O₂ and PaO₂ is pO₂ in sampled arterial blood. PAO₂ = [(FiO₂ x (P_atm − P_H2O) − (PaCO₂/respiratory quotient)], where FiO₂ is the fraction of O₂ in inspired air; P_atm is atmospheric pressure; P_H2O is the partial pressure of H₂O in inspired air; PaCO₂ is pCO₂ in arterial blood; and respiratory quotient (RQ) is the ratio of CO₂ eliminated/O₂ consumed. We took FiO₂ of room-air to be 21% = 0.21, P_atm to be 760 mmHg, and P_H2O to be 47 mmHg [76]. We took the RQ value of our adult male rats to be 0.9 [90,91].

2.4. Antinociception assessment by Tail-Flick Latency Assay

The antinociceptive actions elicited by intravenous injections of vehicle, L-cysteine and L-CYSee and those elicited by the subsequent injection of morphine, were determined by tail-flick latencies (TFL) via a Tail-Flick Analgesia Meter (IITC Life Science Inc., USA) as detailed previously [68-77,92-94]. The process involved a minor degree of manual restraint when positioning the tail to apply a thermal beam sufficient to induce a latency of tail withdrawal of approximately 3.0 sec. Baseline TFL was tested in all rats prior to any drug administration (−20 min timepoint). One group of rats (82.3 ± 0.4 days of age; 333 ± 3 g body weight; n = 9) then received a bolus IV injection of vehicle (saline, 100 μL/100 g body weight). A second group (83.3 ± 0.4 days of age; 335 ± 3 g body weight; n = 9) received a bolus injection of L-CYSee (500 μmol/kg, IV). And a third group (83.1 ± 0.3 days of age; 332 ± 3 g body weight; n = 6) received a bolus injection of L-cysteine (500 μmol/kg, IV). TFL was tested in the three groups 10 and 20 min after the injection (i.e., at the −10 min and 0 min timepoints, respectively). At + 20 min post-injection, the rats got an injection of morphine (10 mg/kg, IV) and TFL was tested 20-, 40-, 60-, 90-, 120,- 150-, 180-min post-injection. In another study, one group of rats (83.7 ± 0.5 days of age; 339 ± 3 g body weight; n = 9) received an injection of vehicle (saline, 100 μL/100 g body weight), and a second group (83.4 ± 0.4 days of age; 335 ± 3 g body weight; n = 9) received an injection of L-CYSee (500 μmol/kg, IV) and then both groups received an IV injection of a 5 mg/kg dose of morphine and TFL was tested as above. TFL data are presented as actual TFL (sec), and as maximum possible effect (%MPE) determined by the formula, %MPE = [(post-injection TFL − baseline TFL)/(12 − baseline TFL)] x 100 [68-73,92-95].

2.5. Antinociception assessment by Hind-Paw Withdrawal Assay

The antinociceptive actions of morphine, vehicle, L-cysteine, and L-CYSee were assessed by hot-plate (hind-paw withdrawal) latency (HPL) assay using Hargreaves’s method [95] as detailed previously [76,77]. Briefly, HPL in response to a thermal stimulus was assessed using a radiant heat source (IITC, CA, USA) aimed at the planter surface of the left hind-paw. This did not require restraint of the rat while positioning the thermal stimulus sufficient to produce paw withdrawal from the floor of the hot-plate in about 20 s prior to test drug administration (cut-off latency was 40 s to avoid tissue damage). Baseline HPL was tested in all 3 groups (−20 min timepoint). One group of rats (84.0 ± 0.6 days of age; 339 ± 3 g body weight; n = 9) received an IV injection of vehicle (saline, 100 μL/100 g body weight). A second group (83.7 ± 0.5 days of age; 337 ± 3 g body weight; n = 9) received L-cysteine (500 μmol/kg, IV). And a third group (84.2 ± 0.6 days of age; 340 ± 3 g body weight; n = 9) received L-CYSee (500 μmol/kg, IV). HPL was tested 10 and 20 min later (−10 min and 0 min timepoints). At 20 min post-injection (time-point 0 min), all rats received an injection of morphine (10 mg/kg, IV) and HPL was tested 20, 40, 60, 90, 120, 180, 210, 240, 360 and 480 min post-injection. The data are shown as HPL (sec) and as %MPE = [(post-injection HPL − baseline HPL)/(20 − baseline HPL)] x 100.

2.6. Sedation as determined by the modified righting reflex test

This study evaluated the effects of injections of vehicle, L-cysteine (500 μmol/kg, IV), and L-CYSee (500 μmol/kg, IV) on the duration of the effects of morphine (10 mg/kg, IV) on the modified righting reflex test. Each rat was placed in an open container to evaluate the loss of the modified righting reflex. Administration of morphine caused the rats to assume numerous types of postures, including being motionless and sprawled out on their stomach on the chamber floor, lying motionless on their side, and splayed out on their stomach with the head up against the chamber wall. The duration of the morphine sedation was taken as the time interval from the time of morphine injection to full recovery of the righting reflex (i.e., when rats attained and maintained a normal posture on all 4 legs [96-98] as described previously [72-76]. One group of rats (82.0 ± 0.5 days of age; 332 ± 3 g body weight; n = 9) received morphine (10 mg/kg, IV) and after 15 min, vehicle (saline). A second group (81.0 ± 0.5 days of age; 330 ± 3 g body weight; n = 9) received morphine (10 mg/kg, IV) and after 15 min, L-CYSee (500 μmol/kg, IV). A third group (82.2 ± 0.4 days of age; 340 ± 3 g body weight; n = 6) received morphine (10 mg/kg, IV) and after 15 min, L-cysteine (500 μmol/kg, IV). A fourth group (82.0 ± 0.5 days of age; 336 ± 3 g body weight; n = 6) received morphine (10 mg/kg, IV) and after 15 min, L-SERee (500 μmol/kg, IV). And a fifth group (83.1 ± 0.6 days of age; 335 ± 3 g body weight; n = 6) received morphine (10 mg/kg, IV) and after 15 min, L-serine (500 μmol/kg, IV).

2.7. Data Analyses

The directly recorded and arithmetically-derived parameters (i.e., 1 min bins) were taken for statistical analyses. Pre-drug 1 min bins excluded occasional marked deviations from resting values due to abrupt movements by the rats, such as scratching. The exclusions ensured accurate determination of baseline values. All data were evaluated by one-way and two-way ANOVA followed by Bonferroni corrections for multiple comparisons between means using the error mean square terms from each ANOVA analysis [99-101] as detailed previously [72-76]. A p < 0.05 value denoted the initial level of significance that was modified according to the number of comparisons between means as described by Wallenstein et al. [99]. The modified t-statistic is t = (mean group 1 − mean group 2)/[s x (1/n₁ + 1/n₂)^1/2] where s² = the mean square within groups term from the ANOVA (i.e., the square root of this value is used in the modified t-statistic formula), and n₁ and n₂2 are the number of rats in each group being compared. Based on an elementary (Bonferroni’s) inequality, a conservative critical value for modified t-statistics was obtained from tables of t-distribution using a significance level of P/m, where m is the number of comparisons between groups to be performed [102]. The degrees of freedom are those of the mean square for within group variation from the ANOVA table. In the majority of situations, the critical Bonferroni value cannot be found in conventional tables of the t- distribution, but can be approximated from tables of the normal curve by t = z + (z + z³)/4 n, with n degrees of freedom and z is the critical normal curve value for P/m [99-101]. Wallenstein et al. [99] first demonstrated that the Bonferroni procedure is preferable for general use since it is easy to apply, has the widest range of applications, and it provides critical values that are lower than those of other procedures when the number of comparisons can be limited, and will be slightly larger than those of other procedures if many comparisons are made. A value of p < 0.05 was taken as the initial level of statistical significance [99-101] and statistical analyses were performed with the aid of GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).

3. Results

3.1. Effects of L-CYSee and L-cysteine on the ventilatory responses to morphine

The ages, body weights, and baseline (Pre) ventilatory parameter values before injection of vehicle (saline) or L-CYSee are shown in Supplemental Table 4. There were no-between group differences for any parameters (p > 0.05, for all comparisons). Total (cumulative) responses elicited by morphine (10 mg/kg, IV) during the 15 min prior to injection of vehicle or L-CYSee are shown in Supplemental Table 5. Morphine decreased Freq, TV, MV, PIF, PIF/PEF, relaxation time, and inspiratory and expiratory drives, that were accompanied by increases in Ti, Te, Ti/Te, EIP, EEP, EF₅₀, expiratory delay (Te-RT), apneic pause, NEBI, and NEBI/Freq, but minimal changes in PEF. Many responses were present at the post-15 min timepoint namely, the decreases in Freq, TV, MV, PIF, PIF/PEF, relaxation time, and inspiratory and expiratory drives, and substantial increases in Ti, Ti/Te, EIP, EF₅₀, expiratory delay, and apneic pause. The changes in Te, EEP, PEF or NEBI and NEBI/Freq were not present at the post 15-min timepoint.

The minute-by-minute Freq, TV, and MV values recorded before, after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle or L-CYSee (500 μmol/kg, IV), and during a H-H challenge (Air OFF) and return to room-air (Air ON) are summarized in Fig. 1. As seen in Panel A, morphine elicited a transient increase in Freq that was followed by a sustained decrease in Freq that was present at the +15 min timepoint. when the first injection of vehicle or L-CYSee was given. The two injections of vehicle given 15 min apart elicited negligible responses. The first injection of L-CYSee elicited a sustained increase in Freq compared to pre-morphine values. The second injection of L-CYSee increased Freq to above pre-morphine values that gradually resolved over the following 60 min recording. As seen in Panel B, morphine elicited a sustained decrease in TV that was present when injections of vehicle or L-CYSee were given. Neither injection of vehicle changed TV, which gradually recovered toward pre-morphine levels by the end of the recording period. The first injection of L-CYSee elicited a prompt and sustained reversal of the effects of morphine on TV. The second injection further elevated TV and was present 60 min later. As seen in Panel C, the changes in Freq and TV translated into morphine-induced decreases in MV, and a prompt and a sustained reversal in MV back to baseline values with first injection of L-CYSee, and and increase in MV values to above baseline after the second injection of L-CYSee. As seen in Panels A-C, the H-H challenge caused gradual and substantial increases in Freq, TV, and MV. Temporal increases were parallel in vehicle- and L-CYSee-treated rats, but higher values were apparent in L-CYSee rats likely because pre-H-H values were greater than in vehicle rats. Upon return to room-air, Freq remained elevated, whereas TV returned toward pre-morphine levels in vehicle-treated rats, such that MV returned toward pre-morphine levels. Freq values in L-CYSee-treated rats fell below pre-morphine levels, whereas TV fell, but stayed above pre-morphine levels, such that MV fell toward pre-morphine levels.

Fig. 1.

Frequency of breathing (Panel A), tidal volume (Panel B) and minute ventilation (Panel C) in freely-moving rats before (Pre), after the injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 2 ×500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). Data are presented as mean ± SEM. There were 6 rats in each group.

As seen in Fig. 2 (Panel A), morphine elicited a transient decrease followed by a sustained increase in Ti. The two injections of vehicle elicited minor changes in Ti, whereas the first, and especially the second injection of L-CYSee elicited substantial and sustained decreases in Ti. As seen in Panel B, morphine elicited a transient increase in Te that was followed by a sustained decrease in Te. The two injections of vehicle elicited minimal responses, whereas the second injection of L-CYSee, in particular, elicited a substantial and sustained decrease in Te. As seen in Panel C, the respiratory quotient (Ti/Te) fell immediately after both injections of L-CYSee, but then quickly rose substantially within 4 min after either injection. The two injections of vehicle produced negligible responses. Moreover, the second injection of L-CYSee caused a smaller sustained increase in Ti/Te compared to the first injection. As also seen in Panels A-C, the subsequent H-H challenge caused parallel decreases in Ti in both vehicle- and L-CYSee-treated rats (Panel A), whereas the H-H challenge maintained the decreases in Te in both groups (Panel B), such that Ti/Te gradually fell to equivalent levels (Panel C). Upon returning to room-air, Ti dipped noticeably in vehicle-treated rats, but rose to pre-morphine values in the L-CYSee-treated rats (Panel A). In contrast, Te rose to pre-morphine levels in both groups of rats (Panel B) that taken together resulted in Ti/Te returning toward pre-morphine values in both groups (Panel C). As seen in Fig. 3 (Panel A), the injection of morphine elicited a sustained increase in EIP that was maintained after both injections of vehicle. The first injection of L-CYSee elicited a transient decrease in EIP that increased to near morphine values after approximately 7 min post application, whereas the second injection of L-CYSee elicited a profound and long-lasting decrease in EIP. As seen in Panel B, morphine elicited a transient increase in EEP followed by a sustained decrease in the vehicle-treated rats. The two injections of L-CYSee produced somewhat further decreases in EEP. As seen in Panel A, the H-H challenge elicited gradual, yet pronounced, decreases in EIP in both groups of rats with values falling to similar levels, whereas EEP remained at very low values in both groups. Upon return to room-air, EIP remained steady in both groups (Panel A). The return to room-air in vehicle-treated rats was accompanied by a large and sustained rise in EEP that was absent in the L-CYSee-treated rats.

Fig. 2.

Inspiratory time (Panel A), expiratory time (Panel B), and inspiratory time/expiratory time (Ti/Te) (Panel C) in freely-moving rats before (Pre), after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 2 ×500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). Data are presented as mean ± SEM. There were 6 rats each group.

Fig. 3.

End inspiratory pause (Panel A) and end expiratory pause (Panel B) in freely-moving rats before (Pre), after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 2 × 500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). Data are presented as mean ± SEM. There were 6 rats in each group.

As seen in Fig. 4 (Panel A), morphine elicited a transient increase, followed by a sustained decrease, in PIF that did not change in vehicle-treated rats. L-CYSee elicited pronounced and sustained increases in PIF, with the first injection reversing the effects of morphine, and the second injection elevating PIF to values considerably above pre-morphine values. Morphine elicited a transient increase in PEF that had returned to near baseline values when vehicle or L-CYSee were given (Panel B). L-CYSee elicited sustained increases in PEF to levels above pre-morphine values, whereas vehicle injection showed no change from morphine. The changes in PIF and PEF resulted in morphine eliciting sustained decreases in PIF/PEF, with L-CYSee causing sustained increases toward, but not reaching, pre-morphine values (Panel C). As seen in Panel D, the injection of morphine elicited a transient increase, followed by a gradual and sustained elevation in EF₅₀ in vehicle-treated rats. L-CYSee elicited pronounced and sustained increases in EF₅₀. As also seen in Panels A-D, the H-H challenge caused pronounced and parallel changes in PIF, PEF and EF₅₀ in both groups of rats, whereas the decreased PIF/PEF remained steady throughout the H-H challenge in the vehicle- and L-CYSee-treated rats. Upon return to room-air, PIF, PEF and EF₅₀ fell toward pre-morphine levels in both groups, whereas PIF/PEF rose toward pre-morphine levels in both groups. As seen in Panels A and B of Supplemental Fig. 2, morphine elicited a transient decrease followed by a gradual and sustained drop in relaxation time and expiratory delay (Te-RT). Both injections of L-CYSee elicited a transient increase, and then further decrease in these parameters, with the second injection causing a greater decrease then the first injection. As seen in Panel C, morphine elicited an increase in apneic pause that was minimally affected by L-CYSee. As also seen in Supplemental Fig. 2 Panels A-C, the H-H challenge minimally affected the depressed relaxation time, expiratory delay or elevated apneic pause in either group. Upon return to room-air, relaxation time (Panel A) and expiratory delay (Panel B) returned to near pre-morphine values, whereas the elevated apneic pause values did not change (Panel C).

Fig. 4.

Peak inspiratory flow (Panel A), peak expiratory flow (Panel B), peak inspiratory flow/peak expiratory flow (PIF/PEF) (Panel C), and EF₅₀ (Panel D) in freely-moving rats before (Pre), after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 2 ×500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). The data are presented as mean ± SEM. There were 6 rats in each group.

As shown in Fig. 5, the injection of morphine elicited a prompt and sustained decrease in inspiratory drive (Panel A), and a transient reduction in expiratory drive (Panel B) that did not recover to pre-morphine values. The two injections of L-CYSee elicited pronounced and sustained increases in inspiratory drive, with the second injection causing values to approach pre-morphine levels, and expiratory drive, with the second injection to achieve levels greatly above pre-morphine levels. The H-H challenge caused pronounced increases in inspiratory and expiratory drives that were parallel in the vehicle-injected and L-CYSee-injected rats. The values returned toward pre-morphine values on return to room-air in the vehicle- and L-CYSee-treated rats. As shown in Fig. 6, the injection of morphine elicited a transient increase in NEBI (Panel A) and NEBI/Freq (Panel B) that was followed by sustained decreases in both parameters. Both injections of L-CYSee elicited minor responses and did not alter the time-course of morphine-induced changes in either NEBI or NEBI/Freq. The H-H challenge minimally affected NEBI or NEBI/Freq, whereas there was a large increase in both parameters upon return to room-air in the vehicle-treated rats, but not a noticeably large increase in L-CYSee-treated rats.

Fig. 5.

Inspiratory drive (TV/Ti) (Panel A) and expiratory drive (TV/Te) (Panel B) in freely-moving rats before (Pre), after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethyl ester (L-CYSee, 2 ×500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 6.

Non-eupneic breathing index (NEBI) (Panel A) and NEBI corrected for the frequency of breathing (Panel B) in freely-moving rats before (Pre), after injection of morphine (10 mg/kg, IV), after subsequent injections of vehicle (saline) or L-cysteine ethyl ester (L-CYSee, 2 ×500 μmol/kg, IV), and during a hypoxic-hypercapnic challenge (Air OFF) and return to room-air (Air ON). The data are presented as mean ± SEM. There were 6 rats in each group.

The total (cumulative) changes in ventilatory parameters recorded over the 15 min period after the first injection of vehicle or L-CYSee (expressed as %change from Pre-values) are shown in Fig. 7. The first injection of L-CYSee caused a sustained reversal of the deleterious effects of morphine on Freq, TV, MV, PIF, inspiratory (InspD) and expiratory (ExpD) drives, whereas it augmented the increases in PEF and EF₅₀, and decrease in EEP. The first injection of L-CYSee did not alter the morphine-induced increases in Ti, Ti/Te, EIP, expiratory delay (Te-RT), or apneic pause ((Te/RT)− 1), or the decreases in Te, PIF/PEF, relaxation time (RT), NEBI or NEBI/Freq. The total changes in ventilatory parameters over the 15 min period after the second injection of vehicle or L-CYSee (%change from Pre-values) are seen in Fig. 8. The second injection of L-CYSee caused a sustained reversal of the morphine-induced decreases in Freq, TV, MV, PIF, and inspiratory and expiratory drives, a decrease of morphine-induced elevations in Ti and EIP, an augmentation of morphine-induced increases in PEF and EF₅₀, an augmentation of morphine-induced decreases in EEP, NEBI and NEBI/Freq, but no changes in morphine-induced increases in Ti/Te, expiratory delay (Te-RT), or apneic pause ((Te/RT)− 1), or decreases in Te, PIF/PEF, and relaxation time (RT). The total changes in ventilatory parameters recorded over the 60 min period of H-H challenge (%change from Pre-values) are shown in Fig. 9. The increases in Freq, TV, MV, EF₅₀, apneic pause ((Te/RT)− 1), and inspiratory and expiratory drives were greater in L-CYSee-injected rats compared to vehicle-treated rats. The increases in Ti and EIP seen during H-H challenge in vehicle-treated rats were abolished by L-CYSee. The decreases in Te, EEP, PIF/PEF, NEBI, and NEBI/Freq, and increases in Ti/Te, PIF, PEF, and expiratory delay (Te-RT) seen during H-H challenge in vehicle-treated rats were not affected by L-CYSee. The total changes over the 30 min period upon return to room-air (expressed as %change from Pre-values) are shown in Fig. 10. The increases in Freq, EEP, NEBI and NEBI/Freq were reduced or reversed in L-CYSee-injected rats compared to vehicle-treated rats, whereas the increases in TV and expiratory delay (Te-RT) were greater in L-CYSee-injected rats. The changes in all other parameters were similar in both groups. In contrast to L-CYSee, L-SERee (500 μmol/kg, IV) did not modify the effects of morphine (10 mg/kg, IV) on Freq, TV or MV, or changes in these parameters during H-H challenge or upon return to room-air (Supplemental Fig. 3).

Fig. 7.

Total cumulative changes (expressed as the %change from Pre-values) in ventilatory parameters that occurred over the 15 min period following the first injection of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV). The data were analyzed by one-way ANOVA. There were 6 rats in each group. The data are presented as mean ± SEM. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

Fig. 8.

Total cumulative changes (expressed as the %change from Pre-values) in ventilatory parameters that occurred over the 15 min period following the second injection of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV). The data were analyzed by one-way ANOVA. There were 6 rats in each group. The data are presented as mean ± SEM. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

Fig. 9.

Total cumulative changes (expressed as %change from Pre-values) in ventilatory parameters that occurred over the 60 min period of hypoxic-hypercapnic (H-H) challenge in rats that received morphine (10 mg/kg, IV) and two injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV). The data were analyzed by one-way ANOVA. There were 6 rats in each group. The data are presented as mean ± SEM. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

Fig. 10.

Total cumulative changes (expressed as %change from Pre-values) in ventilatory parameters that occurred over the 30 min period following return to room-air after a hypoxic-hypercapnic (H-H) challenge in rats that received morphine (10 mg/kg, IV) and two injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV). The data were analyzed by one-way ANOVA. There were 6 rats in each group. The data are presented as mean ± SEM. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

3.2. Effects of L-CYSee or L-cysteine on morphine changes in ABG chemistry and A-a gradient

ABG values (pH, pCO₂, pO₂, sO₂) before and after injection of morphine (10 mg/kg, IV), and then injection of vehicle, L-cysteine (500 μmol/kg, IV) or L-CYSee (500 μmol/kg, IV) in three groups of rats are summarized in Fig. 11. Values denoted M15 to M75 are timepoints post-morphine injection, and values denoted D0 to D60 reflect the timepoints after injection of vehicle, L-cysteine or L-CYSee. As seen in Panel A, morphine elicited substantial and equivalent decreases in arterial blood pH in the three groups of rats. L-CYSee, but not L-cysteine, elicited a prompt and sustained reversal of the acidosis. As seen in Panel B, morphine produced a substantial and similar increase in pCO₂ in the three treatment groups. L-CYSee, but not L-cysteine, caused a sustained reversal of the increase in arterial pCO₂. As seen in Panels C and D, morphine elicited sustained decreases in pO₂ and sO₂, respectively in the three treatment groups of rats, and L-CYSee, but not L-cysteine, elicited a prompt and sustained reversal of the hypoxemia. As shown in Fig. 12, morphine (10 mg/kg, IV) produced substantial and equivalent increases in A-a gradient values (i.e., diminished alveolar gas-exchange) in the three groups of rats. L-CYSee (500 μmol/kg, IV), but not L-cysteine (500 μmol/kg, IV), reversed the adverse effects of morphine.

Fig. 11.

Values of pH (Panel A), pCO₂ (Panel B), pO₂ (Panel C) and sO₂ (Panel D) before (Pre) and after injection of morphine (10 mg/kg, IV) in three separate groups of freely-moving rats, followed by injection of vehicle (saline), L-cysteine (500 μmol/kg, IV) or L-cysteine ethyl ester (L-CYSee, 500 μmol/kg, IV). The terms M15, M30, M45, M60 and M75 denote 15, 30, 45, 60 and 75 min after injection of morphine. The terms D0, D15, D30, D45 and D60 denote 0, 15, 30, 45 and 60 min after injection of test compound (i.e., vehicle, L-cysteine or L-CYSee). The data were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons between means. The data are presented as mean ± SEM. There were 9 rats in each group. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

Fig. 12.

Alveolar-arterial (A-a) A-a gradient values before (Pre) and after injection of morphine (10 mg/kg, IV) in three separate groups of freely-moving rats, followed by injection of vehicle (saline), L-cysteine (500 μmol/kg, IV) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV). The terms M15, M30, M45, M60 and M75 denote 15, 30, 45, 60 and 75 min after injection of morphine. The terms D0, D15, D30, D45 and D60 denote 0, 15, 30, 45 and 60 min after injection of test compound (i.e., vehicle, L-cysteine or L-CYSee). The data were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons between means. The data are mean ± SEM. There were 9 rats in each group. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

3.3. Effects of L-CYSee or L-cysteine on the antinociceptive actions of morphine – TFL and HPL

TFLs before and after injection of 5 or 10 mg/kg doses of morphine, and then an injection of vehicle or L-CYSee (500 μmol/kg, IV) are shown in Panel A and Panel B of Fig. 13 and Supplemental Table 6. Changes expressed as maximal possible effect (%MPE) are shown in Panels C and D. The initial analgesic effects of morphine were similar in both groups, but waned more quickly in L-CYSee-injected rats. HPLs before and after injection of 5 or 10 mg/kg doses of morphine, and then an injection of vehicle or L-CYSee (500 μmol/kg, IV) are shown in Panel A and Panel B of Fig. 14 and Supplemental Table 7. The changes expressed as maximal possible effect are shown in Panels C and D, respectively. As with the TFLs, the initial antinociceptive effects of morphine were similar in both groups, but waned more quickly in the L-CYSee-injected rats. As shown in Supplemental Fig. 4, injections of L-cysteine (500 μmol/kg, IV) (Panels A and B), L-SERee (500 μmol/kg, IV) (Panels C and D), or L-serine (500 μmol/kg, IV) (Panels E and F), did not modify the effects of a 5 mg/kg (left panels) or 10 mg/kg (right panels) dose of morphine.

Fig. 13.

Tail-flick latencies following injection of morphine (5 or 10 mg/kg, IV) and then injection of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV) in freely-moving rats (Panels A and B, respectively), and respective changes in tail-flick latency expressed as maximum possible effect (%MPE) in the 5 and 10 mg/kg morphine-treated rats (Panels C and D, respectively). The data were analyzed by two-way ANOVA with Bonferroni corrections for multiple comparisons between means. The data are shown as mean ± SEM. There were 9 rats in each group. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

Fig. 14.

Hot-plate latencies following injection of morphine (5 or 10 mg/kg, IV), and then injections of vehicle (saline) or L-cysteine ethylester (L-CYSee, 500 μmol/kg, IV) in freely-moving rats (Panels A and B, respectively), and respective changes in hot-plate latency expressed as maximum possible effect (%MPE) in the 5 and 10 mg/kg morphine-treated rats (Panels C and D, respectively). The data were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons between means. The data are shown as mean ± SEM. There were 9 rats in each group. *p < 0.05, significant change from Pre-values. ^†p < 0.05, L-CYSee versus vehicle.

3.4. Effects of L-cysteine and L-CYSee and morphine-induced sedation

The behavioral phenomena in the rats that received morphine plus vehicle, L-cysteine or L-CYSee were not different to one another. The injection of morphine elicited a relatively rapid (within 2–3 min) sedative effect in rats that consisted as an almost total loss of mobility and unusual body postures. The full return of the modified righting-reflex in vehicle-treated rats (79.6 ± 7.1 min, n = 9), L-cysteine (500 μmol/kg, IV)-treated rats (67.9 ± 6.4 min, n = 6), and L-CYSee (500 μmol/kg, IV)-treated rats (85.3 ± 7.4 min, n = 9) were similar to one another (p > 0.05, for all between-group comparisons).

4. Discussion

We report here that L-CYSee reverses the effects of morphine on breathing in freely-moving adult male Sprague Dawley rats. The ventilatory responses elicited by the 10 mg/kg dose of morphine were similar to those we reported previously [9,72-74,76,82,84]. In vehicle-treated rats, morphine elicited a sustained reduction in frequency accompanied by sustained increase in Ti (longer inspiratory duration), but a less impactful decrease in Te (shorter expiratory duration). Additionally, morphine elicited a long-lasting increase in EIP, but a transient increase in EEP, that was followed by a sustained decrease. The ability of morphine to lengthen Tim while only marginally affecting Te is well-described [103-105], as is shortening of Te, and modulating EIP and EEP [76,78-80,82,84]. Mechanisms of action of opioids in brain structures, such as the nucleus tractus solitarius [106-109], pre-Bötzinger complex [110,111], and parabrachial nucleus/Kölliker-Fuse nucleus [110-113], with respect to eliciting different effects on inspiratory and expiratory timing, have been explored, and data suggests that the qualitative and quantitative effects of opioids on Ti and Te are dose-dependent [80,114]. However, the mechanisms and sites of action by which opioids, such as morphine and fentanyl, exert their differential effects on EIP and EEP have received limited study and there is no definitive understanding [80,11]. Previous reports from studies in anesthetized rats and in vitro cell preparations, suggest that a reduction in carotid body chemoreceptor reflex activity plays an important role in the mechanism by which morphine reduces Freq of breathing. Additionally, our lab has previously reported that the ability of morphine (10 mg/kg, IV) to depress Freq was enhanced in unanesthetized rats with bilateral carotid sinus nerve transection [81], suggesting that morphine augments carotid body chemoreflex activity, which defends against the adverse effects of morphine. As reported [76,78-80,82,84], morphine also elicited long-lasting decreases TV, MV, PIF, inspiratory and expiratory drives, relaxation time and expiratory delay (Te-RT), accompanied by sustained increases in EF₅₀, apneic pause, and NEBI. This multi-factorial series of ventilatory effects caused by morphine, points to an important role of opioid-receptor signaling processes in the control of breathing.

The first novel finding in this study was that systemic injection of L-CYSee overcame the adverse actions of morphine on breathing in freely-moving rats. Overall, L-CYSee reversed the inhibitory actions of morphine on Freq, TV, MV, Ti, EIP, PIF, and inspiratory and expiratory drives, whereas it augmented morphine-induced decreases in Te and EEP, and augmented morphine-induced increases in PEF and EF₅₀, but minimally affected morphine-induced reduction in NEBI and NEBI/Freq. The first injection of L-CYSee reversed most of the effects of morphine to pre-morphine levels, and the second injection often augmented the effects on ventilation produced by the first injection of L-CYSee. These findings add to our understanding of the pharmacology of L-CYSee and its related analogue, L-CYSme, including (1) oxidation to disulfide di(m)ethylesters [115], (2) interaction with alpha-lipoic acid to form mixed disulfides [116], (3) changes in physicomechanical properties of dipalmitoyl-phosphatidyl-choline in the plasma membrane [117], (4) hydrogen bonding between L-thiol esters to chloride ions [118], (5) dynamic interactions with myoglobin [119], (6) one and two electron-dependent reduction of cytochrome C leading to enhanced respiratory chain activity [120], (7) modulation of cardiac intracellular Ca²⁺ concentrations in response to mechanical stress [121], (8) formation of [122] or one-electron breakdown of S-nitrosothiols [123,124], (9) regulation of enzymatic activities of aspartate/alanine aminotransferases [125], (10) isomerization of 9-cis-retinoic acid [126], (11) capacity to be substrates of peroxidases [127-130], (12) disassembly-reassembly of [2Fe-2S] clusters in the redox-regulated transcription factor SoxR [131], (13) potentiation of glucose-stimulated release of insulin [132], (14) diminish the viscosity of mucus [133] and thus the capacity to enter lungs tissues [134-136], and (15) to diminish pulmonary edema [137]. We have not established how L-CYSee exerts its beneficial actions against morphine-induced OIRD. The lack of immediate effects of L-CYSee toward morphine-induced antinociception and sedation suggests that unlike opioid receptor antagonists, such as naloxone, L-CYSee does not act as a competitive or allosteric inhibitor of opioid receptors. Our findings that neither L-cysteine nor L-SERee were able to modulate any of the actions of morphine suggests that the entry of L-CYSee into neurons/cells, and subsequent thiol-dependent chemistry, are fundamentally important. We predict that upon entry into cells, L-CYSee may itself interact directly with cell-signaling proteins mediating OIRD. It is also possible that intracellular de-esterification of L-CYSee to L-cysteine leads to alterations in intracellular redox status [138-140], and generation of bioactive factors, including S-nitrosothiols [53-59], hydrogen sulfide, via sequential actions of L-cysteine aminotransferase and cystathionine γ-lyase in peripheral and central tissues [45-47], including the carotid bodies [48], and cysteine-sulfenic, -sulfinic and -sulfonic acid derivatives [49-52]. The lack of activity of L-cysteine when injected may be because morphine reduces L-cysteine uptake into neurons/cells via the inhibition of excitatory amino acid transporter type 3 [10,11]. It is not known if morphine can block other L-cysteine up-take systems [141-144], including excitatory amino acid transporters 1 and 2 [144-147], large neutral amino acid transporters [148-151], band 3 protein-anion transport system [141,152], and high affinity Na⁺-dependent glutamate transporters [153].

The pharmacokinetic distribution of L-CYSee in neural and other cell entities may be due to its entry and subsequent effect on morphine-induced OIRD. Autoradiographic studies have shown that intravenous injection (10 mg/kg, 100 μCi/kg) of ³⁵S-L-CYSee [154] or ³⁵S-L-CYSme [155] resulted in their rapid appearance in blood cells and tissues. At 5 min, the densities of ³⁵S-L-CYSee and ³⁵S-L-CYSme were highest within the lungs and kidneys with high densities also observed within the chest-wall, intestines and brain (only ³⁵S-L-CYSee studied in the brain). Low levels of ³⁵S-L-CYSee and ³⁵S-L-CYSme were observed in plasma, most likely due to the rapid dispersal of L-thiolesters into tissues. After 60 min, the densities of ³⁵S-L-CYSee and ³⁵S-L-CYSme and were very high within lungs, chest-wall muscles, liver, kidneys, and intestines, but diminished in the brain, although several regions, such as the brainstem, still displayed very strong labeling. Additionally, these studies showed that intravenous injection of ³⁵S-L-cysteine (10 mg/kg, 100 μCi/kg) resulted in intense labeling in liver and kidneys, but minimal labeling in brain, lungs or chest-wall muscle [154]. Taken together, it is likely that L-CYSee enters central (e.g., brainstem) and peripheral (e. g., carotid bodies and chest-wall muscle) structures that mediate the effects of morphine on breathing [156-159] to overcome signaling events responsible for OIRD and disturbances in ABG chemistry. The first injections of L-CYSee or L-CYSme [73] reversed the adverse effects of morphine on breathing, and the second injections elicited pronounced and sustained series of effects, including elevations in Freq, TV and MV. These pronounced responses contrast to our observations with D-CYSee [74] in that the first injection caused an immediate and sustained reversal of the adverse effects of morphine, however the second injection elicited only minor additional responses. As such, it would seem that L-CYSee and L-CYSme activate functional proteins and/or enter into metabolic pathways that are not accessible to D-CYSee. The findings from this study with L-CYSee add to our evidence regarding the efficacy of reduced and disulfide D,L-thiolesters [71-77] to prevent and/or reverse the adverse actions of morphine on ventilatory processes. The abilities of L-CYSee and L-CYSme to overcome OIRD may involve the intracellular generation of their S-nitrosylated forms, namely, SNO-L-CYSee and SNO-L-CYSme, since morphine- or fentanyl-induced effects on ventilatory parameters, ABG chemistry, and A-a gradient were substantially diminished in rats that were receiving continuous intravenous infusions of SNO-L-cysteine [68,69], and the adverse effects of fentanyl were exaggerated after administration of a nitric oxide synthase inhibitor [67]. It is well-known that SNO-L-cysteine controls numerous intracellular signaling cascades [56-66] and, as mentioned above, including those involved in diminishing the effects of opioids on breathing [68,69]. The effectiveness of bolus injections of L-CYSee to reverse the deleterious actions of morphine on breathing, and A-a gradient, and ABG chemistry in freely-moving rats, compliments our evidence that intravenous infusion of L-CYSee also markedly diminishes the deleterious effects of morphine [72].

4.1. L-CYSee reverses morphine-induced changes in ABG chemistry and A-a gradient

Consistent with our previous reports [9,70-78], the injection of morphine (10 mg/kg, IV) elicited sustained changes in ABG chemistry in unanesthetized adult male Sprague Dawley rats that included decreases in arterial blood pH, pO₂ and sO₂, and increases in pCO₂, all changes consistent with hypoventilation. These changes in ABG chemistry may have also resulted from a diminished alveolar gas exchange since morphine increased A-a gradient [9,70-78]. The morphine-induced increase in A-a gradient may involve collapse of alveoli (atelectasis), due to hypoventilation, and adverse effects on surfactant function and alveolar fluid clearance. Therefore, the end-result of a therapy to overcome OIRD must be restoration of ABG chemistry, and so the second, and perhaps most vital set of findings of this study was that a single injection of L-CYSee (but not L-cysteine or L-SERee) elicited a sustained reversal of the deleterious effects of morphine on A-a gradient and ABG chemistry. Previous studies reveal that the ability of L-CYSee to overcome the effects of morphine on breathing are not compromised by untoward effects of L-CYSee on upper airways and/or gas-exchange in isoflurane-anesthetized morphine-treated rats [71]. Although the abilities of L-CYSee and other D,L-thiolesters to overcome the actions of morphine on breathing and alveolar gas-exchange may involve direct increase of intracellular reducing redox equivalents, the redox reducing cell-permeable L-thiolester, N-acetyl-L-cysteine methylester (L-NACme) [160], minimally affected OIRD [76]. Rapid conversion of L-NACme to L-cysteine in cells [134] would argue against the beneficial effects of L-CYSee against OIRD being due to entry of L-cysteine into intracellular pathways or provision of reducing equivalents in cells. Rather, these findings suggest that the efficacy of L-CYSee may involve the intracellular actions of the thiolester moiety and direct modification of cell-signaling processes responsible for OIRD. The ability of the free-radical/superoxide anion scavenger, Tempol, to markedly reduce OIRD elicited by morphine [78] and fentanyl [79] demonstrates the importance of redox process in the actions of opioids. The effectiveness of injections of L-CYSee to reverse the adverse effects of morphine on ABG chemistry and A-a gradient in freely-moving rats compliments our previous evidence that the ventilatory-depressant effects of morphine are markedly diminished in conscious rats receiving intravenous infusion of L-CYSee [72] and reversed by injection of L-CYSee in isoflurane-anesthetized rats with a tracheostomy [71], which was was required because the combination of morphine and L-CYSee was deleterious to upper airway caliber. Taken together, our findings that L-CYSee reverses morphine-induced changes in ABG chemistry and A-a gradient support the need for studies with L-CYSee in pre-clinical, large animal models of OIRD, such as dogs [161,162] or goats [163,164], that bridge potential clinical trials in humans.

4.2. L-CYSee-induced changes in morphine-induced antinociception

Intravenous injections of 5 or 10 mg/kg doses of morphine elicited robust antinociceptive effects in unanesthetized rats of more than 180 min and 240 min in duration, respectively, as measured by TFL and HPL assays [74,76]. The TFL and HPL assays were used to more-fully examine the effects of L-CYSee on the spinal and supraspinal mechanisms by which morphine causes analgesia [68-79]. The injection of the 500 μmol/kg dose of L-CYSee in naïve rats increased TFL and HPL at 10 min, but not 20 min, post-injection. Since no behavioral responses were observed upon injection, or thereafter, it is possible that the increased TFL and HPL may have been a short-lived antinociceptive response. The morphine-induced antinociception at the 150 min and 180 min timepoints were reduced in L-CYSee-injected rats. The 500 μmol/kg dose of L-cysteine reduced TFL and HPL at the 10 min post-injection time, whereas L-serine and L-SERee did not. The changes in nociception elicited by microinjections of L- and D-cysteine and L- and D-cystine into the hindpaw of rats [165-170] have been studied insensory cell bodies in dorsal root ganglia [171] and thalamus [172,173]. L-cysteine was pronociceptive after micro-injections, whereas the disulfide, L-cystine, is antinociceptive [165-173]. For example, intra-dermal injections of L-cysteine in the peripheral receptive field (ventral side) of a hind paw elicited a dose/time-dependent hyperalgesia in rats that was prevented by blocking voltage-dependent T-type Ca²⁺ channels [168]. Moreover, L-cysteine caused hyperalgesia when microinjected into the thalamus by activation of CaV_3.2, the major molecular substrate for T-type Ca²⁺ channel redox regulation [172,173]. The finding that L-cysteine transiently decreased TFL and HPL, whereas L-CYSee transiently increased TFL and HPL, suggest that the putative algesia elicited by L-cysteine is due to extracellular actions on plasma membrane proteins, whereas the putative analgesic elicited by L-CYSee may involve intracellular actions. The findings that the initial analgesic actions of morphine were not reduced by L-CYSee clearly suggests that the L-thiolester did not block opioid receptors in the sites that elicit the antinociceptive actions of morphine [174,175]. The slightly reduced duration of morphine antinociception in L-CYSee-injected rats suggests that the L-thiol ester affects intracellular processes that countermand the antinociceptive actions of the opioid. It is possible that L-CYSee exerts its antinociceptive actions as the L-thiolester moiety itself, but that these effects are gradually counterbalanced by de-esterification of L-CYSee to L-cysteine intracellularly, which overcomes morphine analgesia by activation of voltage-dependent T-type Ca²⁺ channels [168,174,175]. S-nitroso-L-cysteine diminishes T-type Ca²⁺ channel activity upon injection into the thalamus of rats [172,173,176], and thus s-nitrosylation of L-CYSee to SNO-L-CYSee upon entry into neural nociceptive-antinociceptive pathways may remove the algesic actions of L-cysteine while directly promoting analgesia.

4.3. Effects of morphine and L-CYSee on responses to H-H challenge and upon return to room-air

We demonstrated that the rebreathing method to challenge freely-moving rats with a gradually increasing hypoxic-hypercapnic (H-H) environment elicits pronounced temporally-related increases in frequency, tidal volume, minute ventilation, and many other ventilatory parameters, and that canonical changes in these parameters occurs upon return to room-air [84]. The ventilatory responses to H-H challenge reached greater values (e.g., Freq, TV, MV, PIF, PEF, EF₅₀, and inspiratory and expiratory drives), or lower values (e.g., Ti) in the morphine-treated rats that had received injections of L-CYSee, largely due to the differences in resting values prior to H-H challenge. It is imporant to note that the values observed in the morphine-treated rats that had recieved injections of L-CYSee changed in parallel fashion to the morphine-treated rats that had recieved injections of vehicle. Other parameters (i.e., Te, EIP, EEP, relaxation time, expiratory delay, apneic pause, NEBI, and NEBI/Freq) reached similar endpoints in both groups. A major novel finding of this study was that the return to room-air responses were markedly different in the morphine-treated rats that had received L-CYSee than vehicle. The substantial increases in Freq present at the end of the H-H challenge rose even further upon return to room-air in the vehicle-treated rats, whereas Freq fell to below pre-morphine levels in the L-CYSee-treated rats. The substantial decreases in Ti present at the end of the H-H challenge fell further in the vehicle-treated rats, whereas it rose to pre-morphine levels upon return to room-air in L-CYSee-treated rats. In contrast, Te simply rose to pre-morphine values in both groups. And the elevated levels of TV, MV, PIF, PEF, EF₅₀, and inspiratory and expiratory drives fell similarly in both groups. The key findings were that the substantial increases in EEP, NEBI and NEBI/Freq observed in vehicle-treated rats were virtually absent in those that received L-CYSee. As such, L-CYSee prevented the adverse effects of morphine on transition from expiration to inspiration, and the incidence of non-eupneic breathing episodes. The mechanisms for these effects of L-CYSee are not known, but are likely to be beneficial in a variety of disease states in which obstructive apneas are a concern, including subjects using opioids and other drugs that affect breathing [1-8,84].

We have not tested whether lower doses of L-CYSee overcome OIRD. Lower doses may reverse OIRD and have less effect on the duration of morphine antinociception. Since fentanyl is playing a major role in the current opioid crisis [5-8], it is vital to determine whether L-CYSee can overcome the adverse actions of this opioid. Additionally, we need to determine whether L-CYSee is able to prevent/reverse OIRD in female rats since opioids exert qualitatively/quantitatively different effects on ventilatory systems of females compared to males [177,178]. Data is lacking regarding the subcellular-molecular mechanisms by which L-CYSee modulates OIRD. Putative mechanisms are (1) direct binding to L,D-cysteine binding protein, and/or myristoylated alanine-rich C-kinase substrate [179], (2) interruption of opioid receptor-β-arrestin-coupled cell signal transduction pathways that do not impact G-protein-dependent analgesic actions of morphine [180,181], and/or (3) potential conversion of L-CYSee to S-nitroso-L-CYSee [182] or S-nitroso-L-cysteine [183,184], by S-nitrosylation of the sulfur atom via processes requiring nitric oxide synthase. To test if administration of L-CYSee forms S-nitrosothiols, we are examining whether intravenous injections of L-CYSee increases production of S-nitrosylated species in blood and tissues via the use of an ultra-sensitive capacitive sensor developed by [185], and whether L-CYSee increases expression of NADPH diaphorase, which visualizes free S-nitrosothiols and S-nitrosylated proteins in aldehyde-treated tissue [55]. Previous studies have shown that S-nitrosothiols, such as S-nitroso-L-cysteine and S-nitro-so-L-glutathione, play important roles in ventilatory control processes in the brainstem, red blood cells, and numerous peripheral structures, including the carotid bodies [64,65,186-189]. Of relevance are findings that morphine- and fentanyl-induced OIRD and disturbances in ABG chemistry were markedly diminished in rats that were receiving an intravenous infusion of S-nitroso-L-cysteine [68,69]. The effectiveness of S-nitroso-L-cysteine adds to our knowledge about the ability of L-S-nitrosothiols to regulate cardiorespiratory control systems [53,54,61-67,186-189], and we are currently employing our sensor technology [185] and NADPH diaphorase histochemical method [55] to determine if systemic injections of L-CYSee generate S-nitrosothiols in the carotid bodies and major cardiorespiratory control structures in the brain. To overcome our lack of information about the pharmacokinetics of L-CYSee we are using liquid chromatography-mass spectrometry [190] to determine the temporal distribution of L-CYSee in the blood, and peripheral and central structures relevant to expression of OIRD, including medial prefrontal cortex, brainstem and hypothalamus. To extend our understanding of the effects of L-CYSee, we are testing whether it ameliorates the effects that morphine exerts on ventilatory responses to hypoxic gas exposures [191]. Relevant to all studies in non-human species, the most important concern is whether the efficacies of OIRD reversal agents in rats will translate into therapies that reverse OIRD in humans. Many such OIRD reversal agents showing efficacy in rats lacked effect in human clinical trials [3-5]. A true limitation is our lack of understanding of the mechanism by which L-CYSee reduces the duration of the antinociceptive effects of morphine. The potential mechanisms of action of L-CYSee described above provide us with starting points to explore the mechanisms by which these antinociceptive effects of L-CYSee are exerted.

In summary, L-CYSee overcame the adverse effects of morphine on breathing, A-a gradient and ABG chemistry. In addition, L-CYSee did not affect the duration of morphine sedation, but did slightly diminish the duration of antinociception. Since L-cysteine and L-SERee were inactive, L-CYSee may overcome the adverse actions of morphine on breathing by mechanisms dependent on intracellular thiol-dependent processes. With respect to L-CYSee shortening morphine analgesia, L-CYSee may be converted to compounds that reverse the analgesia or act as algesics. Whether the formation of thiol-morphine conjugates [192] or ability of L-CYSee to promote enzymatic conversion of morphine to major metabolites, such morphine-3-glucuronide [193], needs to be addressed. The ability of L-CYSee to enter neurons or astrocytes in the brain is likely involved in reversing the effects of morphine on breathing, although peripherally-restricted opioid receptor antagonists, such as naloxone methiodide, also reduce the analgesic and cardiorespiratory effects, but not the sedative actions of opioids [80,194,195]. The present findings with L-CYSee add to our knowledge about the efficacy of L,D-thiolesters [70-78] and the free-radical/superoxide anion scavenger, Tempol [79], against morphine- or fentanyl-induced OIRD. With respect to possible contribution to the field, our evidence that L-CYSee reverses the effects of morphine on ventilatory parameters, A-a gradient, and ABG chemistry, adds to our growing body of knowledge about the abilities that L, D-thiolesters, such as L-CYSee [71,72], D-CYSme [73], D-CYSee [74, 75], D-cystine di(m)ethylester [75,76], L-GSHee [77], and the S-nitrosothiols, such as S-nitroso-L-cysteine [68,69] and L-NAC [70] have toattenuate the adverse effects of morphine or fentanyl on breathing and alveolar gas-exchange. A concern with L-CYSee and L-CYSme [73] and to a lesser extent, D-CYSee [74] and D-cystine di(m)ethylester [76], is the decrease in duration of morphine analgesia. This would result in needing to give morphine more frequently in subjects needing pain relief, but would not be problematic in unintended overdose. If the efficacy of L-CYSee translates to humans, it would be feasible to give it as a stand-alone OIRD reversal agent or with opioid receptor antagonists to overcome OIRD. L,D-thiolesters could be given in place of naloxone when maintaining analgesia is essential, such as during and/or after surgery, and when reversal of OIRD is needed in opioid-dependent subjects in which opioid receptor antagonists precipitate withdrawal. L-CYSee is subject to de-esterification to L-cysteine in blood and tissues by plasma carboxylesterases [41-44], thereby reducing the efficacy of the L-thiolester. In the future, we are planning studies with an esterase inhibitor, bis(4-nitrophenyl) phosphate [196,197], which is effective upon systemic injection [198], to see whether it enhances the efficacy and duration of action of L-CYSee and other D,L-thiolesters.

Abbreviations:

L-CYSee

L-cysteine ethylester

L-CYSme

L-cysteine methylester

D-CYSee

D-cysteine ethylester

L-GSHee

L-glutathione ethylester

D-CYSdiee

D-cystine diethylester

D-CYSdime

D-cystine dimethylester

EAA

excitatory amino acid transporter

BK channel

large conductance Ca²⁺-activated K⁺ channel

Freq

frequency of breathing

TV

tidal volume

MV

minute ventilation

Ti

inspiratory time

Te

expiratory time

EIP

end inspiratory pause

EEP

end expiratory pause

PIF

peak inspiratory flow

PEF

peak expiratory flow

EF₅₀

expiratory flow at 50% expired TV

RT

relaxation time

NEBI

non-eupneic breathing index

ABG

arterial blood-gas chemistry

A-a gradient

Alveolar-arterial gradient.

TFL

tail-flick latency

HPL

hind-paw withdrawal latency

Data availability

Data will be made available on request.