Eliane Boudinot

Different effects of μ and δ opiate agonists on respiration

Abstract The involvement of different opiate receptor subtypes in opiate-induced respiratory depression was studied in the unanaesthetized rat. Synthetic opioid agonists, specific for μ or δ receptors, were administered intraperitoneally in freely moving rats while respiratory parameters were recorded by means of the whole body plethysmographic method. TRIMU-4 (Tyr-D-Ala-Gly-NH-CH(CH 3 )-CH 2 -CH(CH 3 ) 2 ), a specific agonist of the μ receptor, reduced the tidal volume and did not change the respiratory frequency. DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a relatively specific agonist of the δ receptor subtype, reduced respiratory frequency and was significantly less effective on tidal volume than was TRIMU-4. It is concluded that the respiratory depression occurring after the administration of opiates in clinical practice is a dual complementary effect involving μ and δ receptors.

Respiratory survival mechanisms in acetylcholinesterase knockout mouse

Cholinergic neurotransmission ensures muscle contraction and plays a role in the regulation of respiratory pattern in the brainstem. Inactivation of acetylcholinesterase (AChE) by organophosphates produces respiratory failure but AChE knockout mice survive to adulthood. Respiratory adaptation mechanisms which ensure survival of these mice were examined in vivo by whole body plethysmography and in vitro in the neonatal isolated brainstem preparation. AChE−/− mice presented no AChE activity but unaffected butyrylcholinesterase (BChE) activity. In vivo, bambuterol (50–500 µg/kg s.c.) decreased BChE activity peripherally but not in brain tissue and induced apnea and death in adult and neonate AChE−/− mice without affecting littermate AChE+/+ and +/− animals. In vitro, bath‐applied bambuterol (1–100 µm) and tetraisopropylpyrophosphoramide (10–100 µm) decreased BChE activity in the brainstem but did not perturb central respiratory activity recorded from spinal nerve rootlets. In vitro, the cholinergic agonists muscarine (50–100 µm) and nicotine (0.5–10 µm) induced tonic activity in respiratory motoneurons and increased the frequency of inspiratory bursts in AChE+/+ and +/− animals. These effects were greatly attenuated in AChE−/− animals. The results suggest that, in mice lacking AChE, (i) BChE becomes essential for survival peripherally but plays no critical role in central rhythm‐generating structures and (ii) a major adaptive mechanism for respiratory survival is the down‐regulated response of central respiratory‐related neurons and motoneurons to muscarinic and nicotinic agonists.

Respiratory function in adult mice lacking the µ-opioid receptor: role of δ-receptors

Mice lacking the µ‐opioid receptor (MOR) provide a unique model to determine whether opioid receptors are functionally interactive. Recent results have shown that respiratory depression produced by δ‐opioid receptor agonists is suppressed in mice lacking the µ‐opioid receptor. Here we investigated the involvement of µ‐ and δ‐opioid receptors in the control of ventilation and µ/δ receptor interactions in brainstem rhythm‐generating structures. Unrestrained MOR–/– and wild‐type mice showed similar ventilatory patterns at rest and similar chemosensory responses to hyperoxia (100% O2), hypoxia (10% O2) or hypercapnia (5%CO2−95%O2). Blockade of δ‐opioid receptors with naltrindole affected neither the ventilatory patterns nor the ventilatory responses to hypoxia in MOR–/– and wild‐type mice. In‐vitro, respiratory neurons were recorded in the pre‐Bötzinger complex of thick brainstem slices of MOR–/– and wild‐type young adult mice. Respiratory frequency was not significantly different between these two groups. The δ2 receptor agonist deltorphin II (0.1–1.0 µm) decreased respiratory frequency in both groups whereas doses of the δ1 receptor agonist enkephalin[D‐Pen2,5] (0.1–1.0 µm) which were ineffective in wild‐type mice significantly decreased respiratory frequency in MOR–/– mice. We conclude that deletion of the µ‐opioid receptor gene has no significant effect on ensuing respiratory rhythm generation, ventilatory pattern, or chemosensory control. In MOR–/– mice, the loss of respiratory‐depressant effects of δ2‐opioid receptor agonists previously observed in vivo does not result from a blunted response of δ receptors in brainstem rhythm‐generating structures. These structures show an unaltered response to δ2‐receptor agonists and an augmented response to δ1‐receptor agonists.

Differentiation of two respiratory areas in the cat medulla using kainic acid

Kainic acid (KA) was used to destroy neuronal perikarya in different areas of the brainstem. Single KA microinjections were performed in 30 anaesthetized, vagotomized, artificially ventilated cats. Consequences were studied on the phrenic nerve activity (PNA) and blood pressure. We observed changes of the PNA unrelated to blood pressure alteration. Destruction of the dorsal respiratory area (DRA) including the nucleus tractus solitarius at the obex level produced a 40% decrease of the PNA frequency. Destruction restricted to the lateral part of the ventral respiratory area (VRA1) including the ambiguus nucleus induced a 60% decrease of the integrated PNA amplitude followed by a 40% increase of PNA frequency. These latter effects were also observed after destruction inside the infra solitary reticular formation (ISRF). No effect was observed after destruction in other brain structures. We concluded that ISRF and VRA1 form a single ventral bulbar respiratory area. This area controls respiration in a way different from that of the dorsal respiratory area (DRA).

Effects of acetylcholinesterase and butyrylcholinesterase inhibition on breathing in mice adapted or not to reduced acetylcholinesterase

We investigated the contributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition to the respiratory dysfunction produced by organophosphates in mice which were adapted or not to low AChE activity. Effects of acute selective inhibition of AChE and BChE on ventilation measured by whole-body plethysmography were compared in mice with either normal AChE activity (wild-type), or mice adapted to a null AChE activity (homozygotes for AChE gene deletion) or adapted to an intermediate level of activity (heterozygotes). In wild-type mice acute reduction of AChE by Huperzine A (1 mg/kg) to the level found in asymptomatic heterozygotes, induced tremors but no respiratory depression, whereas the same dose of Huperzine in heterozygote animals further reduced AChE activity, increased tidal volume (V(T)) and decreased breathing frequency (f(R)). A lethal dose of Huperzine in wild-type mice augmented these respiratory effects, but was ineffective in homozygotes. BChE inhibition by bambuterol was ineffective in wild-type mice and heterozygotes, decreased V(T) in homozygotes adapted to null AChE activity but increased V(T) in wild-type mice acutely treated with Huperzine, also aggravating the cholinergic syndrome. We conclude that: (1) Huperzine does not perturb respiration at a dose inhibiting 40% of AChE, and at a lethal dose does not affect any other enzyme important for respiration; (2) Respiratory function is more sensitive to anticholinesterases in heterozygotes than in wild-type mice; (3) BChE may play distinct roles in respiratory function, because its inhibition has opposite effects on tidal volume depending on whether the mouse has adapted to null AChE or whether AChE has been lowered acutely; (4) BChE inhibition may contribute to the respiratory toxicity of organophosphates.

Increased ventilation and CO2 chemosensitivity in acetylcholinesterase knockout mice

To investigate the effects of a permanent excess of acetylcholine (AChE) on respiration, breathing and chemosensitivity were analyzed from birth to adulthood in mice lacking the AChE gene (AChE-/-), in heterozygotes, and in control wild-type (AChE+/+) littermates. Breathing at rest and ventilatory responses to brief exposures to hypoxia (10% O2) and hypercapnia (3-5% CO2) were measured by whole-body plethysmography. At rest AChE-/- mice show larger tidal volumes (VT, + 96% in adults), overall ventilation (VE, + 70%), and mean inspiratory flow (+270%) than wild-type mice, with no change in breathing frequency (fR). AChE-/- mice have a slightly blunted response to hypoxia, but increased VE and fR responses to hypercapnia. Heterozygous animals present no consistent alterations of breathing at rest and chemosensitivity is normal. Adult AChE-/- mice have an increased VE/VO2 and a marginally higher normalized VO2. The results suggest that the hyperventilation and altered chemosensitivity in AChE-/- mice largely reflect alterations of central respiratory control.

Localization of chemosensitive structures in the isolated brainstem of adult guinea‐pig.

1. Central respiratory chemosensitivity has been intensively examined but some questions remain unsolved; namely, what is the nature of the stimulus (fixed acid and/or CO2) and where is the site of brainstem chemosensitivity (near the ventral medullary surface or structures deeper within the brainstem)? To examine these questions, we used the in vitro isolated brainstem of adult guinea‐pig perfused independently through the basilar artery and the bath. 2. Respiratory motor output was recorded with a suction electrode from cranial hypoglossal (XII) roots. Changes in pH and CO2 in the Krebs perfusate were made by changing either the bicarbonate concentration or the PCO2 saturating the Krebs solution. 3. Changes in basilar artery perfusate consisting of (i) an acidifying increase in PCO2 (hypercapnic acidic Krebs solution), (ii) an increase in PCO2 with no change in pH (hypercapnic Krebs solution), or (iii) a decrease in pH with no change in PCO2 (acidic Krebs solution) evoked increases in respiratory frequency and a concomitant decrease in inspiratory burst amplitude. 4. Bath superfusion with hypercapnic acidic Krebs solution increased the inspiratory burst amplitude with no effect on respiratory burst frequency. 5. Bath superfusion with hypercapnic non‐acidic Krebs solution increased the inspiratory burst amplitude and decreased the respiratory frequency, while normocapnic acidic Krebs solution increased the respiratory frequency with no change in burst amplitude. 6. These results show that respiratory responses to changes in CO2 and pH depend upon the sites of action. While a CO2 increase or a pH decrease affected the respiratory frequency in the deep brainstem structures (perfused through the basilar artery), CO2 respiratory chemosensitivity at the ventral surface could be differentiated from the hydrogen ion chemosensitivity. This suggests that different mechanisms mediated respiratory responses when deep versus superficial brainstem structures were stimulated.

Ventilatory pattern and chemosensitivity in M1 and M3 muscarinic receptor knockout mice

Acetylcholine (ACh) acting through muscarinic receptors is thought to be involved in the control of breathing, notably in central and peripheral chemosensory afferents and in regulations related to sleep-wake states. By using whole-body plethysmography, we compared baseline breathing at rest and ventilatory responses to acute exposure (5 min) to moderate hypoxia (10% O(2)) and hypercapnia (3 and 5% CO(2)) in mice lacking either the M(1) or the M(3) muscarinic receptor, and in wild-type matched controls. M(1) knockout mice showed normal minute ventilation (V(E)) but elevated tidal volume (V(T)) at rest, and normal chemosensory ventilatory responses to hypoxia and hypercapnia. M(3) knockout mice had elevated V(E) and V(T) at rest, a reduced V(T) response slope to hypercapnia, and blunted V(E) and frequency responses to hypoxia. The results suggest that M(1) and M(3) muscarinic receptors play significant roles in the regulation of tidal volume at rest and that the afferent pathway originating from peripheral chemoreceptors involves M(3) receptors.

Respiratory network remains functional in a mature guinea pig brainstem isolated in vitro

SummaryWe previously developed a perfused isolated brainstem preparation in the adult guinea pig (Morin-Surun and Denavit-Saubie 1989a) which permitted us to describe several types of rhythmic neuronal discharge. In the present study, we demonstrate that nearly all the periodic neuronal activity we recorded in the ventral respiratory areas were directly related to the respiratory-like periodic output of the hypoglossal nerve. This respiratory-like activity lasted several hours only when the brainstem was perfused by the basilar artery. This shows the necessity of the intraarterial perfusion to preserve a functional respiratory network. Analysis of the characteristics of hypoglossal respiratory-like activity shows that (1) two types of respiratory rhythms can be recorded; one with long respiratory phases (inspiratory and expiratory) and one with short respiratory phases. Depending on the preparation, either type occurs alone or intermingled with the other. (2) The shape of the inspiratory-like activity can change throughout the recording period while the periodicity remains stable. This preparation generates a respiratory rhythm and enables us to dissociate the different mechanisms involved in respiratory neurogenesis.

Effects of the potent analgesic enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan on respiration

&NA; We investigated whether the enkephalin‐catabolizing enzyme inhibitors RB101 and kelatorphan, which have been shown to be potent analgesics, depress respiration as do opioid analgesics. Ventilation was measured in cats and rodents by the barometric method, in the awake state and during anesthesia. Tissue distribution of the inhibitors was either generalized (RB101, 40–160 mg/kg i.p.), largely restricted by the blood–brain barrier to the periphery (kelatorphan, 0.7–20 mg/kg i.v.), or restricted to the brainstem (i.c.v. injection of RB101 in the fourth ventricle). RB101 did not affect ventilation in any condition tested, and large doses of kelatorphan produced a naloxone‐reversible increase in ventilation and breathing frequency. Thus endogenous opioids released during conditions of normal ventilation do not exert any depressant neuromodulatory effect on this function, even when their extracellular concentrations are increased by peptidase inhibitors. The differential effect of these inhibitors on ventilation and nociception is discussed. We conclude that kelatorphan and RB101 are devoid of respiratory‐depressant effects and might be interesting pharmacological alternatives to morphine and other opioid agonists.