Fentanyl activates opposing opioid and non-opioid receptor systems that control breathing.

Fentanyl elicits profound disturbances in ventilatory control processes in humans and experimental animals. The traditional viewpoint with respect to fentanyl-induced respiratory depression is that once the effects on the frequency of breathing (Freq), tidal volume (TV), and minute ventilation (MV = Freq × TV) are resolved, then depression of breathing is no longer a concern. The results of the present study challenge this concept with findings, as they reveal that while the apparent inhibitory effects of fentanyl (75 µg/kg, IV) on Freq, TV, and MV in adult male rats were fully resolved within 15 min, many other fentanyl-induced responses were in full effect, including opposing effects on respiratory timing parameters. For example, although the effects on Freq were resolved at 15 min, inspiratory duration (Ti) and end inspiratory pause (EIP) were elevated, whereas expiratory duration (Te) and end expiratory pause (EEP) were diminished. Since the effects of fentanyl on TV had subsided fully at 15 min, it would be expected that the administration of an opioid receptor (OR) antagonist would have minimal effects if the effects of fentanyl on this and other parameters had resolved. We now report that the intravenous injection of a 1.0 mg/kg dose of the peripherally restricted OR antagonist, methyl-naloxone (naloxone methiodide, NLXmi), did not elicit arousal but elicited some relatively minor changes in Freq, TV, MV, Te, and EEP but pronounced changes in Ti and EIP. In contrast, the injection of a 2.5 mg/kg dose of NLXmi elicited pronounced arousal and dramatic changes in many variables, including Freq, TV, and MV, which were not associated with increases in non-apneic breathing events such as apneas. The two compelling conclusions from this study are as follows: 1) the blockade of central ORs produced by the 2.5 mg/kg dose of NLXmi elicits pronounced increases in Freq, TV, and MV in rats in which the effects of fentanyl had apparently resolved, and 2) it is apparent that fentanyl had induced the activation of two systems with counter-balancing effects on Freq and TV: one being an opioid receptor inhibitory system and the other being a non-OR excitatory system.

L-NAC and L-NAC methyl ester prevent and overcome physical dependence to fentanyl in male rats.

N-acetyl-L-cysteine (L-NAC) is a proposed therapeutic for opioid use disorder. This study determined whether co-injections of L-NAC (500 µmol/kg, IV) or its highly cell-penetrant analogue, L-NAC methyl ester (L-NACme, 500 µmol/kg, IV), prevent acquisition of acute physical dependence induced by twice-daily injections of fentanyl (125 µg/kg, IV), and overcome acquired dependence to these injections in freely-moving male Sprague Dawley rats. The injection of the opioid receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IV), elicited a series of withdrawal phenomena (i.e. behavioral and cardiorespiratory responses, hypothermia and body weight loss) in rats that received 5 or 10 injections of fentanyl and similar numbers of vehicle co-injections. With respect to the development of dependence, the NLX-precipitated withdrawal phenomena were reduced in rats that received had co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme. In regard to overcoming established dependence, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) were reduced in rats that had received co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme beginning with injection 6 of fentanyl. This study provides compelling evidence that co-injections of L-NAC and L-NACme prevent the acquisition of physical dependence and overcome acquired dependence to fentanyl in male rats. The higher efficacy of L-NACme is likely due to its greater cell penetrability in brain regions mediating dependence to fentanyl and interaction with intracellular signaling cascades, including redox-dependent processes, responsible for the acquisition of physical dependence to fentanyl.

The Reducing Agent Dithiothreitol Modulates the Ventilatory Responses That Occur in Freely Moving Rats during and following a Hypoxic-Hypercapnic Challenge.

The present study examined the hypothesis that changes in the oxidation-reduction state of thiol residues in functional proteins play a major role in the expression of the ventilatory responses in conscious rats that occur during a hypoxic-hypercapnic (HH) gas challenge and upon return to room air. A HH gas challenge in vehicle-treated rats elicited robust and sustained increases in minute volume (via increases in frequency of breathing and tidal volume), peak inspiratory and expiratory flows, and inspiratory and expiratory drives while minimally affecting the non-eupneic breathing index (NEBI). The HH-induced increases in these parameters, except for frequency of breathing, were substantially diminished in rats pre-treated with the potent and lipophilic disulfide-reducing agent, L,D-dithiothreitol (100 µmol/kg, IV). The ventilatory responses that occurred upon return to room air were also substantially different in dithiothreitol-treated rats. In contrast, pre-treatment with a substantially higher dose (500 µmol/kg, IV) of the lipophilic congener of the monosulfide, N-acetyl-L-cysteine methyl ester (L-NACme), only minimally affected the expression of the above-mentioned ventilatory responses that occurred during the HH gas challenge or upon return to room air. The effectiveness of dithiothreitol suggests that the oxidation of thiol residues occurs during exposure to a HH gas challenge and that this process plays an essential role in allowing for the expression of the post-HH excitatory phase in breathing. However, this interpretation is contradicted by the lack of effects of L-NACme. This apparent conundrum may be explained by the disulfide structure affording unique functional properties to dithiothreitol in comparison to monosulfides. More specifically, the disulfide structure may give dithiothreitol the ability to alter the conformational state of functional proteins while transferring electrons. It is also possible that dithiothreitol is simply a more efficient reducing agent following systemic injection, although one interpretation of the data is that the effects of dithiothreitol are not due to its reducing ability.

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

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.

L-cysteine ethyl ester prevents and reverses acquired physical dependence on morphine in male Sprague Dawley rats.

The molecular mechanisms underlying the acquisition of addiction/dependence on morphine may result from the ability of the opioid to diminish the transport of L-cysteine into neurons via inhibition of excitatory amino acid transporter 3 (EAA3). The objective of this study was to determine whether the co-administration of the cell-penetrant L-thiol ester, L-cysteine ethyl ester (L-CYSee), would reduce physical dependence on morphine in male Sprague Dawley rats. Injection of the opioid-receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IP), elicited pronounced withdrawal phenomena in rats which received a subcutaneous depot of morphine (150 mg/kg) for 36 h and were receiving a continuous infusion of saline (20 µL/h, IV) via osmotic minipumps for the same 36 h period. The withdrawal phenomena included wet-dog shakes, jumping, rearing, fore-paw licking, 360° circling, writhing, apneas, cardiovascular (pressor and tachycardia) responses, hypothermia, and body weight loss. NLX elicited substantially reduced withdrawal syndrome in rats that received an infusion of L-CYSee (20.8 µmol/kg/h, IV) for 36 h. NLX precipitated a marked withdrawal syndrome in rats that had received subcutaneous depots of morphine (150 mg/kg) for 48 h) and a co-infusion of vehicle. However, the NLX-precipitated withdrawal signs were markedly reduced in morphine (150 mg/kg for 48 h)-treated rats that began receiving an infusion of L-CYSee (20.8 µmol/kg/h, IV) at 36 h. In similar studies to those described previously, neither L-cysteine nor L-serine ethyl ester (both at 20.8 µmol/kg/h, IV) mimicked the effects of L-CYSee. This study demonstrates that 1) L-CYSee attenuates the development of physical dependence on morphine in male rats and 2) prior administration of L-CYSee reverses morphine dependence, most likely by intracellular actions within the brain. The lack of the effect of L-serine ethyl ester (oxygen atom instead of sulfur atom) strongly implicates thiol biochemistry in the efficacy of L-CYSee. Accordingly, L-CYSee and analogs may be a novel class of therapeutics that ameliorate the development of physical dependence on opioids in humans.

Hypoxia releases S-nitrosocysteine from carotid body glomus cells-relevance to expression of the hypoxic ventilatory response.

We have provided indirect pharmacological evidence that hypoxia may trigger release of the S-nitrosothiol, S-nitroso-L-cysteine (L-CSNO), from primary carotid body glomus cells (PGCs) of rats that then activates chemosensory afferents of the carotid sinus nerve to elicit the hypoxic ventilatory response (HVR). The objective of this study was to provide direct evidence, using our capacitive S-nitrosothiol sensor, that L-CSNO is stored and released from PGCs extracted from male Sprague Dawley rat carotid bodies, and thus further pharmacological evidence for the role of S-nitrosothiols in mediating the HVR. Key findings of this study were that 1) lysates of PGCs contained an S-nitrosothiol with physico-chemical properties similar to L-CSNO rather than S-nitroso-L-glutathione (L-GSNO), 2) exposure of PGCs to a hypoxic challenge caused a significant increase in S-nitrosothiol concentrations in the perfusate to levels approaching 100 fM via mechanisms that required extracellular Ca2+, 3) the dose-dependent increases in minute ventilation elicited by arterial injections of L-CSNO and L-GSNO were likely due to activation of small diameter unmyelinated C-fiber carotid body chemoafferents, 4) L-CSNO, but not L-GSNO, responses were markedly reduced in rats receiving continuous infusion (10 µmol/kg/min, IV) of both S-methyl-L-cysteine (L-SMC) and S-ethyl-L-cysteine (L-SEC), 5) ventilatory responses to hypoxic gas challenge (10% O2, 90% N2) were also due to the activation of small diameter unmyelinated C-fiber carotid body chemoafferents, and 6) the HVR was markedly diminished in rats receiving L-SMC plus L-SEC. This data provides evidence that rat PGCs synthesize an S-nitrosothiol with similar properties to L-CSNO that is released in an extracellular Ca2+-dependent manner by hypoxia.

Infrared light elicits endothelium-dependent vasodilation in isolated occipital arteries of the rat via soluble guanylyl cyclase-dependent mechanisms.

The left and right occipital arteries provide blood supply to afferent cell bodies in the ipsilateral nodose and petrosal ganglia. This supply is free of an effective blood-ganglion barrier, so changes in occipital artery blood flow directly affect the access of circulating factors to the afferent cell bodies. The application of infrared (IR) light to modulate neural and other cell processes has yielded information about basic biological processes within tissues and is gaining traction as a potential therapy for a variety of disease processes. To address whether IR can directly modulate vascular function, we performed wire myography studies to determine the actions of IR on occipital arteries isolated from male Sprague-Dawley rats. Based on our previous research that functionally-important differences exist between occipital artery segments close to their origin at the external carotid artery (ECA) and those closer to the nodose ganglion, the occipital arteries were dissected into two segments, one closer to the ECA and the other closer to the nodose ganglion. Segments were constricted with 5-hydroxytryptamine to a level equal to 50% of the maximal response generated by the application of a high (80 mM) concentration of K+ ions. The direct application of pulsed IR (1,460 nm) for 5 s produced a rapid vasodilation in occipital arteries that was significantly more pronounced in segments closest to the ECA, although the ECA itself was minimally responsive. The vasodilation remained for a substantial time (at least 120 s) after cessation of IR application. The vasodilation during and following cessation of the IR application was markedly diminished in occipital arteries denuded of the endothelium. In addition, the vasodilation elicited by IR in endothelium-intact occipital arteries was substantially reduced in the presence of a selective inhibitor of the nitric oxide-sensitive guanylate cyclase, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). It appears that IR causes endothelium-dependent, nitric-oxide-mediated vasodilation in the occipital arteries of the rat. The ability of IR to generate rapid and sustained vasodilation may provide new therapeutic approaches for restoring or improving blood flow to targeted tissues.

Male histone deacetylase 6 (HDAC6) knockout mice have enhanced ventilatory responses to hypoxic challenge.

Histone deacetylase 6 (HDAC6) is a class II histone deacetylase that is predominantly localized in the cytoplasm of cells. HDAC6 associates with microtubules, regulating acetylation of tubulin and other proteins. The possibility that HDAC6 participates in hypoxic signaling is supported by evidence that (1) hypoxic gas challenges cause microtubule depolymerization, (2) expression of hypoxia inducible factor alpha (HIF)-1α is regulated by microtubule alterations in response to hypoxia, and (3) inhibition of HDAC6 prevents HIF-1α expression and protects tissue from hypoxic/ischemic insults. The aim of this study was to address whether the absence of HDAC6 alters ventilatory responses during and/or after hypoxic gas challenges (10% O2, 90% N2 for 15 min) in adult male wild-type (WT) C57BL/6 mice and HDAC6 knockout (KO) mice. Key findings were that (1) baseline values for frequency of breathing, tidal volume, inspiratory and expiratory times and end expiratory pause were different between KO mice and WT mice, (2) ventilatory responses during hypoxic challenge were more robust in KO mice than WT mice for parameters including frequency of breathing, minute ventilation, inspiratory and expiratory durations, peak inspiratory and expiratory flows, inspiratory and expiratory drives, and (3) responses upon return to room-air were markedly different in KO mice than WT mice for frequency of breathing, minute ventilation, inspiratory and expiratory durations, end expiratory (but not end inspiratory) pauses, peak inspiratory and expiratory flows, and inspiratory or expiratory drives. These data suggest that HDAC6 may have a fundamentally important role in regulating the neural responses to hypoxia.

Loss of ganglioglomerular nerve input to the carotid body impacts the hypoxic ventilatory response in freely-moving rats.

The carotid bodies are the primary sensors of blood pH, pO2 and pCO2. The ganglioglomerular nerve (GGN) provides post-ganglionic sympathetic nerve input to the carotid bodies, however the physiological relevance of this innervation is still unclear. The main objective of this study was to determine how the absence of the GGN influences the hypoxic ventilatory response in juvenile rats. As such, we determined the ventilatory responses that occur during and following five successive episodes of hypoxic gas challenge (HXC, 10% O2, 90% N2), each separated by 15 min of room-air, in juvenile (P25) sham-operated (SHAM) male Sprague Dawley rats and in those with bilateral transection of the ganglioglomerular nerves (GGNX). The key findings were that 1) resting ventilatory parameters were similar in SHAM and GGNX rats, 2) the initial changes in frequency of breathing, tidal volume, minute ventilation, inspiratory time, peak inspiratory and expiratory flows, and inspiratory and expiratory drives were markedly different in GGNX rats, 3) the initial changes in expiratory time, relaxation time, end inspiratory or expiratory pauses, apneic pause and non-eupneic breathing index (NEBI) were similar in SHAM and GGNX rats, 4) the plateau phases obtained during each HXC were similar in SHAM and GGNX rats, and 5) the ventilatory responses that occurred upon return to room-air were similar in SHAM and GGNX rats. Overall, these changes in ventilation during and following HXC in GGNX rats raises the possibility the loss of GGN input to the carotid bodies effects how primary glomus cells respond to hypoxia and the return to room-air.

Hypercapnic signaling influences hypoxic signaling in the control of breathing in C57BL6 mice.

Interactions between hypoxic and hypercapnic signaling pathways, expressed as ventilatory changes occurring during and following a simultaneous hypoxic-hypercapnic gas challenge (HH-C) have not been determined systematically in mice. This study in unanesthetized male C57BL6 mice addressed the hypothesis that hypoxic (HX) and hypercapnic (HC) signaling events display an array of interactions indicative of coordination by peripheral and central respiratory mechanisms. We evaluated the ventilatory responses elicited by hypoxic (HX-C, 10%, O2, 90% N2), hypercapnic (HC-C, 5% CO2, 21%, O2, 90% N2), and HH-C (10% O2, 5%, CO2, 85% N2) challenges to determine whether ventilatory responses elicited by HH-C were simply additive of responses elicited by HX-C and HC-C, or whether other patterns of interactions existed. Responses elicited by HH-C were additive for tidal volume, minute ventilation and expiratory time, among others. Responses elicited by HH-C were hypoadditive of the HX-C and HC-C responses (i.e., HH-C responses were less than expected by simple addition of HX-C and HC-C responses) for frequency of breathing, inspiratory time and relaxation time, among others. In addition, end-expiratory pause increased during HX-C, but decreased during HC-C and HH-C, therefore showing that HC-C responses influenced the HX-C responses when given simultaneously. Return to room-air responses was additive for tidal volume and minute ventilation, among others, whereas they were hypoadditive for frequency of breathing, inspiratory time, peak inspiratory flow, apneic pause, inspiratory and expiratory drives, and rejection index. These data show that HX-C and HH-C signaling pathways interact with one another in additive and often hypoadditive processes.NEW & NOTEWORTHY We present data showing that the ventilatory responses elicited by a hypoxic gas challenge in male C57BL6 mice are markedly altered by coexposure to hypercapnic gas challenge with hypercapnic responses often dominating the hypoxic responses. These data suggest that hypercapnic signaling processes activated within brainstem regions, such as the retrotrapezoid nuclei, may directly modulate the signaling processes within the nuclei tractus solitarius resulting from hypoxic-induced increase in carotid body chemoreceptor input to these nuclei.

Lipophilic analogues of D-cysteine prevent and reverse physical dependence to fentanyl in male rats.

We examined whether co-injections of the cell-permeant D-cysteine analogues, D-cysteine ethyl ester (D-CYSee) and D-cysteine ethyl amide (D-CYSea), prevent acquisition of physical dependence induced by twice-daily injections of fentanyl, and reverse acquired dependence to these injections in freely-moving male Sprague Dawley rats. Injection of the opioid receptor antagonist, naloxone HCl (NLX, 1.5 mg/kg, IV), elicited a series of withdrawal phenomena that included cardiorespiratory and behavioral responses, and falls in body weight and body temperature, in rats that received 5 or 10 injections of fentanyl (125 µg/kg, IV), and the same number of vehicle co-injections. Regarding the development of physical dependence, the NLX-precipitated withdrawal phenomena were markedly reduced in fentanyl-injected rats that had received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV). Regarding reversal of established dependence to fentanyl, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) was markedly reduced in rats that received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV), starting with injection 6 of fentanyl. This study provides evidence that co-injections of D-CYSee and D-CYSea prevent the acquisition of physical dependence, and reverse acquired dependence to fentanyl in male rats. The lack of effect of D-cysteine suggests that the enhanced cell-penetrability of D-CYSee and D-CYSea into cells, particularly within the brain, is key to their ability to interact with intracellular signaling events involved in acquisition to physical dependence to fentanyl.

Male histone deacetylase 6 (HDAC6) knockout mice have enhanced ventilatory responses to hypoxic challenge.

Histone deacetylase 6 (HDAC6) is a class II histone deacetylase that is predominantly localized in the cytoplasm of cells. HDAC6 associates with microtubules and regulates acetylation of tubulin and other proteins. The possibility that HDAC6 participates in hypoxic signaling is supported by evidence that 1) hypoxic gas challenges cause microtubule depolymerization, 2) expression of hypoxia inducible factor alpha (HIF-1α) is regulated by microtubule alterations in response to hypoxia, and 3) inhibition of HDAC6 prevents HIF-1α expression and protects tissue from hypoxic/ischemic insults. The aim of this study was to address whether the absence of HDAC6 alters ventilatory responses during and/or after hypoxic gas challenge (10% O2, 90% N2 for 15 min) in adult male wildtype (WT) C57BL/6 mice and HDAC6 knock-out (KO) mice. Key findings were that 1) baseline values for frequency of breathing, tidal volume, inspiratory and expiratory times, and end expiratory pause were different between knock-out mice and wildtype mice, 2) ventilatory responses during hypoxic challenge were more robust in KO mice than WT mice for recorded parameters including, frequency of breathing, minute ventilation, inspiratory and expiratory durations, peak inspiratory and expiratory flows, and inspiratory and expiratory drives, and 3) responses upon return to room-air were markedly different in KO compared to WT mice for frequency of breathing, minute ventilation, inspiratory and expiratory durations, end expiratory pause (but not end inspiratory pause), peak inspiratory and expiratory flows, and inspiratory and expiratory drives. These data suggest that HDAC6 may have a fundamentally important role in regulating the hypoxic ventilatory response in mice.

The ventilatory depressant actions but not the antinociceptive effects of morphine are blunted in rats receiving intravenous infusion of L-cysteine ethyl ester.

This study demonstrates that intravenous infusion of the cell-penetrant thiol ester, L-cysteine ethyl ester (L-CYSee), to adult male Sprague-Dawley rats elicited (a) minor alterations in frequency of breathing, expiratory time, tidal volume, minute ventilation, or expiratory drive but pronounced changes in inspiratory time, end-inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50, relaxation time, apneic pause, inspiratory drive and non-eupneic breathing index, (b) minimal changes in arterial blood-gas (ABG) chemistry (pH, pCO2, pO2, SO2) and Alveolar-arterial (A-a) gradient (index of alveolar gas exchange), and (c) minimal changes in antinociception (tail-flick latency). Subsequent injection of morphine (10 mg/kg, IV) elicited markedly smaller effects on the above parameters, ABG chemistry, and A-a gradient in rats receiving L-CYSee, whereas morphine antinociception was not impaired. Infusions of L-cysteine or L-serine ethyl ester (oxygen rather than sulfur moiety), did not affect morphine actions on ABG chemistry or A-a gradient. L-CYSee (250 µmol/kg, IV) injection elicited dramatic changes in ventilatory parameters given 15 min after injection of morphine in rats receiving L-CYSee. Our findings suggest that (a) L-CYSee acts in neurons that drive ventilation, (b) L-CYSee reversal of the adverse actions of morphine on ventilation, ABG chemistry and A-a gradient may be via modulation of intracellular signaling pathways activated by morphine rather than by direct antagonism of opioid receptors since morphine antinociception was not diminished by L-CYSee, and (c) the thiol moiety of L-CYSee is vital to efficacy, (d) intracellular conversion of L-CYSee to an S-nitrosylated form may be part of its mechanism of action.

L-cysteine methyl ester overcomes the deleterious effects of morphine on ventilatory parameters and arterial blood-gas chemistry in unanesthetized rats.

We are developing a series of thiolesters that produce an immediate and sustained reversal of the deleterious effects of opioids, such as morphine and fentanyl, on ventilation without diminishing the antinociceptive effects of these opioids. We report here the effects of systemic injections of L-cysteine methyl ester (L-CYSme) on morphine-induced changes in ventilatory parameters, arterial-blood gas (ABG) chemistry (pH, pCO2, pO2, sO2), Alveolar-arterial (A-a) gradient (i.e., the index of alveolar gas-exchange within the lungs), and antinociception in unanesthetized Sprague Dawley rats. The administration of morphine (10 mg/kg, IV) produced a series of deleterious effects on ventilatory parameters, including sustained decreases in tidal volume, minute ventilation, inspiratory drive and peak inspiratory flow that were accompanied by a sustained increase in end inspiratory pause. A single injection of L-CYSme (500 µmol/kg, IV) produced a rapid and long-lasting reversal of the deleterious effects of morphine on ventilatory parameters, and a second injection of L-CYSme (500 µmol/kg, IV) elicited pronounced increases in ventilatory parameters, such as minute ventilation, to values well above pre-morphine levels. L-CYSme (250 or 500 µmol/kg, IV) also produced an immediate and sustained reversal of the deleterious effects of morphine (10 mg/kg, IV) on arterial blood pH, pCO2, pO2, sO2 and A-a gradient, whereas L-cysteine (500 µmol/kg, IV) itself was inactive. L-CYSme (500 µmol/kg, IV) did not appear to modulate the sedative effects of morphine as measured by righting reflex times, but did diminish the duration, however, not the magnitude of the antinociceptive actions of morphine (5 or 10 mg/kg, IV) as determined in tail-flick latency and hindpaw-withdrawal latency assays. These findings provide evidence that L-CYSme can powerfully overcome the deleterious effects of morphine on breathing and gas-exchange in Sprague Dawley rats while not affecting the sedative or early stage antinociceptive effects of the opioid. The mechanisms by which L-CYSme interferes with the OR-induced signaling pathways that mediate the deleterious effects of morphine on ventilatory performance, and by which L-CYSme diminishes the late stage antinociceptive action of morphine remain to be determined.

S-nitroso-L-cysteine stereoselectively blunts the adverse effects of morphine on breathing and arterial blood gas chemistry while promoting analgesia.

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 µmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO2 and a decrease in pCO2, (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 µmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO2 and maximal sO2 with sustained increases in pCO2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception.

D-Cysteine Ethyl Ester Reverses the Deleterious Effects of Morphine on Breathing and Arterial Blood-Gas Chemistry in Freely-Moving Rats.

Cell-penetrant thiol esters including the disulfides, D-cystine diethyl ester and D-cystine dimethyl ester, and the monosulfide, L-glutathione ethyl ester, prevent and/or reverse the deleterious effects of opioids, such as morphine and fentanyl, on breathing and gas exchange within the lungs of unanesthetized/unrestrained rats without diminishing the antinociceptive or sedative effects of opioids. We describe here the effects of the monosulfide thiol ester, D-cysteine ethyl ester (D-CYSee), on intravenous morphine-induced changes in ventilatory parameters, arterial blood-gas chemistry, alveolar-arterial (A-a) gradient (i.e., index of gas exchange in the lungs), and sedation and antinociception in freely-moving rats. The bolus injection of morphine (10 mg/kg, IV) elicited deleterious effects on breathing, including depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive. Subsequent injections of D-CYSee (2 × 500 µmol/kg, IV, given 15 min apart) elicited an immediate and sustained reversal of these effects of morphine. Morphine (10 mg/kg, IV) also A-a gradient, which caused a mismatch in ventilation perfusion within the lungs, and elicited pronounced changes in arterial blood-gas chemistry, including pronounced decreases in arterial blood pH, pO2 and sO2, and equally pronounced increases in pCO2 (all responses indicative of decreased ventilatory drive). These deleterious effects of morphine were immediately reversed by the injection of a single dose of D-CYSee (500 µmol/kg, IV). Importantly, the sedation and antinociception elicited by morphine (10 mg/kg, IV) were minimally affected by D-CYSee (500 µmol/kg, IV). In contrast, none of the effects of morphine were affected by administration of the parent thiol, D-cysteine (1 or 2 doses of 500 µmol/kg, IV). Taken together, these data suggest that D-CYSee may exert its beneficial effects via entry into cells that mediate the deleterious effects of opioids on breathing and gas exchange. Whether D-CYSee acts as a respiratory stimulant or counteracts the inhibitory actions of µ-opioid receptor activation remains to be determined. In conclusion, D-CYSee and related thiol esters may have clinical potential for the reversal of the adverse effects of opioids on breathing and gas exchange, while largely sparing antinociception and sedation.

S-Nitroso-L-Cysteine Stereoselectively Blunts the Deleterious Effects of Fentanyl on Breathing While Augmenting Antinociception in Freely-Moving Rats.

Endogenous and exogenously administered S-nitrosothiols modulate the activities of central and peripheral systems that control breathing. We have unpublished data showing that the deleterious effects of morphine on arterial blood-gas chemistry (i.e., pH, pCO2, pO2, and sO2) and Alveolar-arterial gradient (i.e., index of gas exchange) were markedly diminished in anesthetized Sprague Dawley rats that received a continuous intravenous infusion of the endogenous S-nitrosothiol, S-nitroso-L-cysteine. The present study extends these findings by showing that unanesthetized adult male Sprague Dawley rats receiving an intravenous infusion of S-nitroso-L-cysteine (100 or 200 nmol/kg/min) markedly diminished the ability of intravenous injections of the potent synthetic opioid, fentanyl (10, 25, and 50 µg/kg), to depress the frequency of breathing, tidal volume, and minute ventilation. Our study also found that the ability of intravenously injected fentanyl (10, 25, and 50 µg/kg) to disturb eupneic breathing, which was measured as a marked increase of the non-eupneic breathing index, was substantially reduced in unanesthetized rats receiving intravenous infusions of S-nitroso-L-cysteine (100 or 200 nmol/kg/min). In contrast, the deleterious effects of fentanyl (10, 25, and 50 µg/kg) on frequency of breathing, tidal volume, minute ventilation and non-eupneic breathing index were fully expressed in rats receiving continuous infusions (200 nmol/kg/min) of the parent amino acid, L-cysteine, or the D-isomer, namely, S-nitroso-D-cysteine. In addition, the antinociceptive actions of the above doses of fentanyl as monitored by the tail-flick latency assay, were enhanced by S-nitroso-L-cysteine, but not L-cysteine or S-nitroso-D-cysteine. Taken together, these findings add to existing knowledge that S-nitroso-L-cysteine stereoselectively modulates the detrimental effects of opioids on breathing, and opens the door for mechanistic studies designed to establish whether the pharmacological actions of S-nitroso-L-cysteine involve signaling processes that include 1) the activation of plasma membrane ion channels and receptors, 2) selective intracellular entry of S-nitroso-L-cysteine, and/or 3) S-nitrosylation events. Whether alterations in the bioavailability and bioactivity of endogenous S-nitroso-L-cysteine is a key factor in determining the potency/efficacy of fentanyl on breathing is an intriguing question.

L-NAC reverses of the adverse effects of fentanyl infusion on ventilation and blood-gas chemistry.

There is an urgent need for development of drugs that are able to reverse the adverse effects of opioids on breathing and arterial blood-gas (ABG) chemistry while preserving opioid analgesia. The present study describes the effects of bolus injections of N-acetyl-L-cysteine (L-NAC, 500 µmol/kg, IV) on ventilatory parameters, ABG chemistry, Alveolar-arterial (A-a) gradient, sedation (righting reflex) and analgesia status (tail-flick latency assay) in unanesthetized adult male Sprague Dawley rats receiving a continuous infusion of fentanyl (1 µg/kg/min, IV). Fentanyl infusion elicited pronounced disturbances in (1) ventilatory parameters (e.g., decreases in frequency of breathing, tidal volume and minute ventilation), (2) ABG chemistry (decreases in pH, pO2, sO2 with increases in pCO2), (3) A-a gradient (increases that were consistent with reduced alveolar gas exchange), and (4) sedation and analgesia. Bolus injections of L-NAC given 60 and 90 min after start of fentanyl infusion elicited rapid and sustained reversal of the deleterious effects of fentanyl infusion on ventilatory parameters and ABG chemistry, whereas they did not affect the sedative or analgesic effects of fentanyl. Systemic L-NAC is approved for human use, and thus our findings raise the possibility that this biologically active thiol may be an effective compound to combat opioid-induced respiratory depression in human subjects.

D-cysteine ethyl ester and D-cystine dimethyl ester reverse the deleterious effects of morphine on arterial blood-gas chemistry and Alveolar-arterial gradient in anesthetized rats.

We determined whether intravenous injections of the membrane-permeable ventilatory stimulants, D-cysteine ethyl ester (ethyl (2 S)- 2-amino-3-sulfanylpropanoate) (D-CYSee) and D-cystine dimethyl ester (methyl (2 S)- 2-amino-3-[[(2 S)- 2-amino-3-methoxy-3-oxopropyl]disulfanyl] propanoate) (D-CYSdime), could overcome the deleterious actions of intravenous morphine on arterial blood pH, pCO2, pO2 and sO2, and Alveolar-arterial (A-a) gradient (i.e., the measure of exchange of gases in the lungs) in Sprague Dawley rats anesthetized with isoflurane. Injection of morphine (2 mg/kg, IV) caused pronounced reductions in pH, pO2 and sO2 accompanied by elevations in pCO2, all which are suggestive of diminished ventilation, and elevations in A-a gradient, which suggests a mismatch of ventilation-perfusion. Subsequent boluses of D-cysteine ethyl ester (2 ×100 µmol/kg, IV) or D-cystine dimethyl ester (2 ×50 µmol/kg, IV) rapidly reversed of the negative actions of morphine on pH, pCO2, pO2 and sO2, and A-a gradient. Similar injections of D-cysteine (2 ×100 µmol/kg, IV) were without effect, whereas injections of D-cystine (2 ×50 µmol/kg, IV) produced a modest reversal. Our data show that D-cysteine ethyl ester and D-cystine dimethyl ester readily overcome the deleterious effects of morphine on arterial blood gas (ABG) chemistry and A-a gradient by mechanisms that may depend upon their ability to rapidly enter cells. As a result of their known ability to enter the brain, lungs, muscles of the chest wall, and most likely the major peripheral chemoreceptors (i.e., carotid bodies), the effects of the thiolesters on changes in ABG chemistry and A-a gradient elicited by morphine likely involve central and peripheral mechanisms. We are employing target prediction methods to identify an array of in vitro and in vivo methods to test potential functional proteins by which D-CYSee and D-CYSdime modulate the effects of morphine on breathing.

Differential immunostaining patterns of transient receptor potential (TRP) ion channels in the rat nodose ganglion.

Vagal afferents regulate numerous physiological functions including arterial blood pressure, heart rate, breathing, and nociception. Cell bodies of vagal afferents reside in the inferior vagal (nodose) ganglia and their stimulation by various means is being considered as a way to regulate cardiorespiratory responses and control pain sensations. Stimulation of the nodose by exposure to infrared light is recently being considered as a precise way to elicit responses. These responses would likely involve the activity of temperature-sensitive membrane-bound channels. While papers have been published to track the expression of these transient receptor potential ion channels (TRPs), further studies are warranted to determine the in situ expression of the endogenous TRP proteins in the nodose ganglia to fully understand their pattern of expression, subcellular locations, and functions in this animal model. TRP ion channels are a superfamily of Na+ /Ca2+ -channels whose members are temperature- and/or mechano-sensitive and therefore represent a potential set of proteins that will be activated directly or indirectly by infrared light. Here, we report the spatial localization of six TRP channels, TRPV1, TRPV4, TRPM3, TRPM8, TRPA1, and TRPC1, from nodose ganglia taken from juvenile male Sprague-Dawley rats. The channels were detected using immunohistology with fluorescent tags on cryosections and imaged using confocal microscopy. All six TRP channels were detected with different levels of intensity in neuronal cell bodies and some were also detected in axonal fibers and blood vessels. The TRP receptors differed in their prevalence, in their patterns of expression, and in subcellular expression/localization. More specifically, TRPV1, TRPV4, TRPA1, TRPM8, TRPC1, and TRPM3 were found in vagal afferent cell bodies with a wide range of immunostaining intensity from neuron to neuron. Immunostaining for TRPV1, TRPV4, and TRPA1 appeared as fine particles scattered throughout the cytoplasm of the cell body. Intense TRPV1 immunostaining was also evident in a subset of axonal fibers. TRPM8 and TRPC1 were expressed in courser particles suggesting different subcellular compartments than for TRPV1. The localization of TRPM3 differed markedly from the other TRP channels with an immunostaining pattern that was localized to the periphery of a subset of cell bodies, whereas a scattering or no immunostaining was detected within the bulk of the cytoplasm. TRPV4 and TRPC1 were also expressed on the walls of blood vessels. The finding that all six TRP channels (representing four subfamilies) were present in the nodose ganglia provides the basis for studies designed to understand the roles of these channels in sensory transmission within vagal afferent fibers and in the responses elicited by exposure of nodose ganglia to infrared light and other stimuli. Depending on the location and functionality of the TRP channels, they may regulate the flux of Na+ /Ca2+ -across the membranes of cell bodies and axons of sensory afferents, efferent (motor) fibers coursing through the ganglia, and in vascular smooth muscle.