Chronic exposure of neonatal rat adrenomedullary chromaffin cells to opioids in vitro blunts both hypoxia and hypercapnia chemosensitivity

Abstract

At birth, rat adrenomedullary chromaffin cells (AMCs) respond directly to asphyxial stressors such as hypoxia and hypercapnia by triggering catecholamine secretion, which is critical for proper transition to extrauterine life. These non-neurogenic responses are suppressed postnatally in parallel with the development of splanchnic innervation, and reappear following denervation of the adult adrenal gland. To test whether neural factors released from the splanchnic nerve may regulate AMC chemosensitivity, we previously showed that nicotinic agonists in utero and in vitro suppressed hypoxia, but not hypercapnia, sensitivity. Here, we considered the potential role of opiate peptides which are also released from the splanchnic nerve and act via postsynaptic μ-, δ- and -opioid receptors. Treatment of neonatal rat AMC cultures for ∼1 week with μ- and/or δ- (but not ) opioid agonists (2 μm) led to a marked suppression of both hypoxia and hypercapnia sensitivity, as measured by K(+) current inhibition and membrane depolarization; co-incubation with naloxone prevented the effects of combined opioids. The suppression of hypoxia sensitivity was attributable to upregulation of K(ATP) current density and the K(ATP) channel subunit Kir6.2, and was reversed by the K(ATP) channel blocker, glibenclamide. By contrast, suppression of hypercapnia sensitivity was associated with down-regulation of two key mediators of CO(2) sensing, i.e. carbonic anhydrase I and II. Collectively, these studies point to a novel role for opioid receptor signalling in the developmental regulation of chromaffin cell chemosensitivity, and suggest that prenatal exposure to opioid drugs could lead to impaired arousal responses in the neonate.

Figure 1. Effects of chronic exposure of neonatal rat AMCs to opioid agonists for ∼1 week in vitro on hypoxia sensitivity

A, in control (untreated) AMCs cultured for ∼1 week, hypoxia (∼ 15 mmHg) typically causes inhibition of outward K⁺ current (top trace) and membrane depolarization (bottom trace); current density (pA/pF) versus voltage (I–V) plot (middle) shows significant inhibition of outward current (P < 0.05) at potentials positive to +10 mV. These responses are markedly reduced or abolished in neonatal AMCs chronically exposed for ∼1 week to either a combination of μ-, δ- and κ-opioid agonists (2 μm) (B), or to either μ- or δ-opioid agonists (2 μm) alone (D and E, respectively). These blunting effects of chronic opioids on the hypoxia-induced responses are prevented during continuous co-incubation with the general opioid receptor antagonist naloxone (2 μm; C). Also, chronic exposure to the κ-opioid agonist (2 μm) alone was ineffective (F). Data are presented as mean ± SEM (n= 11). TTX was present in the extracellular solution. C, control; Hox, hypoxia; W, wash.

Figure 2. Effects of glibenclamide on outward K⁺ current and membrane potential during hypoxia in control vs. opioid-treated neonatal AMCs

In control (untreated) AMCs, glibenclamide (50 μm) enhances the hypoxia-induced inhibition of outward K⁺ current as shown in upper sample traces (steps to + 30 mV) and lower current density vs. voltage (I–V) plot (Aa); significant difference (P < 0.05) between current density in hypoxia (Hox) and hypoxia plus glibenclamide (Hox + Glib) at potentials ≥+30 mV. By contrast, opioid-treated cells failed to respond to hypoxia alone, but when combined with glibenclamide, there was a pronounced inhibition of outward current at positive potentials (> +20 mV), as shown in upper traces and lower I–V plot (Ab). Data are presented as mean ± SEM. (P < 0.05). Under current clamp, the hypoxia-induced membrane depolarization and increased excitability (Ba) was potentiated when glibenclamide was applied to the same control cell (Bb) (n= 11). By contrast, membrane depolarization was absent or weak when hypoxia was applied to opioid-treated cells (Ca, Da), and even a weak hyperpolarization occurred in a few cases (Ea). However, when these same cells were exposed to hypoxia in the presence of glibenclamide, there was a marked positive or depolarizing shift in membrane potential as shown in Cb, Db and Eb, respectively; note the cell Ca that was initially quiescent during hypoxia actually fired action potentials when hypoxia was combined with glibenclamide (Cb). The ‘n’ values for each type of response are shown on the right. C, control; Hox, hypoxia; W, wash; Glib, glibenclamide.

Figure 3. Upregulation of functional K_ATP channels and Kir6.2 subunit in opioid-treated neonatal rat AMCs

The glibenclamide-sensitive difference current density (IK_ATP (pA/pF)) in untreated versus opioid-treated AMCs is plotted against voltage in the I–V plot (A), and during steps to +30 mV (B). Note the significant increase in K_ATP current density in opioid-treated relative to control untreated AMCs (P < 0.05 in B). C, Western blot analysis of K_ATP channel subunit, Kir6.2, in untreated AMCs and in AMCs cultured with combined μ-, δ- and κ-opioid agonists (2 μm) for 7 days. Note increased expression of Kir6.2 during chronic opioid exposure; β-actin was used as an internal control. Values are presented as mean ± SEM of three independent experiments (*P < 0.05).

Figure 4. Effects of chronic opioid exposure on CO₂ sensitivity in neonatal rat AMCs

In control neonatal AMCs cultured for ∼1 week, isohydric hypercapnia (10% CO₂; pH 7.4) typically causes inhibition of outward K⁺ current (top trace) and membrane depolarization (bottom trace); current density (pA/pF) versus voltage (I–V) plot (middle) shows significant inhibition of outward current (P < 0.05) at positive potentials (A). These responses are markedly reduced or abolished in neonatal AMCs chronically exposed for ∼1 week to either a combination of μ-, δ- and κ-opioid agonists (2 μm) (B), or to either μ- or δ-opioid agonists (2 μm) alone (D and E, respectively). These blunting effects of chronic opioids on the CO₂-induced responses are prevented during continuous co-incubation with the general opioid receptor antagonist naloxone (2 μm) (C). Also, chronic exposure to the κ-opioid agonist (2 μm) alone was ineffective (F). Data are presented as mean ± SEM (n= 11). TTX was present in the extracellular solution. C, control; W, wash.

Figure 5. Comparative estimates of the blunting effects of chronic opioids on CO₂-mediated responses in neonatal AMCs

Comparison of the CO₂-induced changes in holding current at −60 mV in control (Aa) versus opioid-treated (Ab) cells; note the significant (*P < 0.05) inward shift in holding current normally seen in control cells during high CO₂ is abolished in opioid-treated cells. The variability in CO₂-induced changes in membrane potential is shown for control cells (Ba, Bb) versus opioid-treated cells (Ca, Cb); data of response frequency for each condition are summarized in D. Note that CO₂-induced action potential firing or spikes (Ba) or subthreshold depolarizations (Bb) occur frequently in untreated (control) cells (D), but is rare in opioid-treated cells (Cb, D). Also, the majority of opioid-treated cells fail to show either CO₂-induced membrane depolarization or spikes, i.e. are non-responsive (Ca, D), in contrast to untreated cells (D).

Figure 6. Effects of chronic opioid exposure on CA enzyme expression in neonatal rat AMCs

A, Western blot analysis showing expression of CA isoforms CAI and CAII in 7-day-old cultures of control untreated AMCs versus AMCs treated with combined μ-, δ- and κ-opioid agonists (2 μm); β-actin was used as an internal control. Note the downregulation in CAI and CAII expression in neonatal AMCs following chronic opioid exposure. Values are represented as mean ± SEM of three independent experiments (*P < 0.05).

Figure 7. Immunofluorescence staining of neonatal rat AMC cultures for μ- and δ-opioid receptor expression

Corresponding phase and fluorescence (FITC) images of cultures showing positive immunostaining of AMCs for μ-opioid receptor (MOR) (A and B, respectively), and for δ-opioid receptor (DOR) (C and D, respectively). Pre-absorption controls with excess antigen (see Methods) confirming staining specificity for each antibody are shown in E–H. The data are representative of three independent experiments for each antibody staining.