βγ G-proteins, but not regulators of G-protein signaling 4, modulate opioid-induced respiratory rate depression

Figures

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  FIGURE 1. Neural mechanisms of neuronal inhibition by MORs and expression of Oprm1 and Tacr1 (the gene encoding neurokinin-1 receptors) in the medulla. (A) Neuronal inhibition by MORs is regulated by G-protein-signaling including α, ß and γ proteins and regulators of G-protein signaling. (B) Microperfusion of the MOR agonist DAMGO directly into the preBötC depressed respiratory rate in anesthetized rats. (C) The location of the microperfusion site was identified with histological lesion made by the microdialysis probe shaft. Note that the probe extends beyond the guide by 0.5 mm. (D) The preBötC and the nucleus tractus solitarius (NTS) expressed Oprm1 (the gene coding for MORs) and Tacr1 (the gene coding for tachykinin-1 or neurokinin-1 receptors) mRNAs (E, F) The density of Oprm1 and Tacr1 were assessed in the preBötC and the NTS (G, H). MOR, µ-opioid receptor. GTP, guanine trisphosphate. GDP, guanine diphosphate. RGS, regulators of G-protein-signaling. GIRK, G-protein-activated inwardly rectifying potassium channels. Dia, diaphragm muscle. aCSF, artificial cerebrospinal fluid. Mean data are presented as means ± SEM. Panel a was created using Biorender.com.

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  FIGURE 2. Inhibition of βγ proteins reverses respiratory rate depression by the MOR agonist DAMGO. (A) DAMGO reduced respiratory rate by about 15.5% when compared to aCSF or vehicle, and this reduction was reversed by gallein at 5 mM, but only partially at 1 mM. Naloxone did not increase respiratory rate beyond the aCSF or gallein values. (B) DAMGO reduced respiratory rate compared to aCSF and gallein (1 mM) partially reversed respiratory rate. Although diaphragm amplitude was reduced by DAMGO, this reduction was not reversed by gallein. (C) DAMGO significantly depressed respiratory rate, an effect completely reversed by gallein at 5 mM. No effects were observed in diaphragm amplitude. aCSF, artificial cerebrospinal fluid. MOR, µ-opioid receptors. Bars show means ± SEM. Each circle represents an individual experiment. * indicate means significantly different with p < 0.05.

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  FIGURE 3. Site of action of DAMGO and gallein overlaps with Oprm1 and Tacr1 expressions. To determine the brainstem region where DAMGO is acting, we correlated the latency for DAMGO to decrease respiratory rate with the distances from the preBötC to the microperfusion sites. The latency in minutes for a 10% decrease from baseline respiratory rate was calculated for each experiment (A). For each corresponding experiment, the distance from the microperfusion site (indicated in green) to the center of the preBötC was calculated (B). The coordinates for the preBötC are 12.4 mm posterior, 6 mm ventral, and 2.0 lateral to Bregma. A significant exponential correlation was found between drug latencies and distances (R2 = 0.8078, p < 0.001, n = 10 experiments) (C). To determine whether other sites in the brainstem may mediate some of the respiratory depression induced by DAMGO, we generated a color map of the brainstem section where each pixel (every 50 µm) corresponds to a coefficient of correlation (blue equals low correlation, whereas red high correlation). For each set of coordinates in the section, the correlation coefficient was calculated using latencies and the distances from the…

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  FIGURE 4. Inhibition of RGS4 has limited impact on respiratory rate depression by DAMGO. (A) Inhibition of RGS4 potentiates MOR inhibition by the ligand DAMGO. (B) Pharmacological inhibition of RGS4 by CCG50014 significantly accentuated respiratory depression by DAMGO perfused in the preBötC compared to DAMGO alone. (C) The combination of DAMGO + CCG50014 (5 µM) induced a stronger respiratory rate depression than DAMGO alone (5 µM). Naloxone partially reversed respiratory rate depression by DAMGO and CCG50014. (D) Diaphragm amplitude was not significantly reduced by DAMGO with or without CCG50014. To determine whether RGS4 inhibition alone affects respiratory rate, we microperfused CCG50014 to the preBötC. CCG50014 (5 µM) decreased significantly respiratory rate (E) but not diaphragm amplitude (F). aCSF, artificial cerebrospinal fluid. MOR, µ-opioid receptors. RGS, regulators of G-protein-signaling. * indicate means significantly different. Circles indicate mean values ±SEM.

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  FIGURE 5. The site of action of DAMGO and CCG50014 overlaps with Oprm1 and Tacr1 expressing neurons. Using the same approach than in the gallein experiments, we determined the sites of drug perfusion and the latency for respiratory rate to decrease in response to DAMGO. Drugs were microperfused in the preBötC region as identified with expression of Tacr1 (gene for neurokinin-1 receptors) (A). A significant correlation was observed between distances from probe site to the centre of the preBötC and the latency for DAMGO to depress respiratory rate by 10% (B). Na, nucleus ambiguus.

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  FIGURE 6. Expression of Oprm1 and Rgs4 mRNAs in the preBötC and NTS. (A) The preBötC and the nucleus tractus solitarius (NTS) expressed Oprm1 (the gene coding for MORs, green) and Rgs4 (the gene coding for regulators of G-protein signaling 4, red) mRNAs. (B) Oprm1 and Rgs4 mRNAs were expressed in the preBötC and most cells expressing Oprm1 also co-expressed Rgs4 (orange). The density of Oprm1-positive cells was moderate compared to the high density of Oprm1 cells in the NTS (C). In the NTS, cells expressed Oprm1 as well as Rgs4 with substantial co-expression. Rgs4, mRNA of regulator of G-protein signaling 4. Oprm1, Gene encoding MORs. NTS, nucleus tractus solitarius. na, nucleus ambiguus. io, inferior olive.