Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms

Abstract

Background: Epidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms.

Methodology/principal findings: Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS)-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies.

Conclusions/significance: These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

Figure 1. Effects of acute intravenous administration of parathion or paraoxon on bronchoconstriction in guinea pigs.

Bronchoconstriction was measured in anesthetized guinea pigs in response to electrical stimulation of both vagus nerves or to intravenous ACh before and after intravenous administration of parathion (1 mg/kg) or paraoxon (100 ng/kg or 100 µg/kg). Baseline bronchoconstrictions were within normal physiological parameters (8–17 mmH2O at 5 Hz, 17–59 mmH2O at 10 Hz, and 51–127 mmH2O at 15 Hz). Parathion (hatched bars) inhibited vagally-induced bronchoconstriction by approximately 50% at all three frequencies (A). In contrast, paraoxon at 100 ng/kg (gray bars) and 100 µg/kg (black bars) acutely increased vagally-induced bronchoconstriction in a frequency-dependent manner (A). Paraoxon at 100 ng/kg did not potentiate ACh-induced bronchoconstriction (B) or significantly inhibit AChE activity (C). However, at 100 µg/kg, paraoxon potentiated ACh-induced bronchoconstriction (B) and inhibited AChE (C). Data are presented as mean ± SE; n = 3–4 guinea pigs (* p<0.05).

Figure 2. Acute intravenous administration of parathion or paraoxon did not significantly potentiate bradycardia in guinea pigs.

Bradycardia was measured in anesthetized guinea pigs in response to electrical stimulation of both vagus nerves or to intravenous ACh before and after intravenous administration of parathion (1 mg/kg) or paraoxon (100 ng/kg or 100 µg/kg). None of the OP treatments had any effect on vagally-induced bradycardia at any of the frequencies tested (A). Baseline bradycardia at 5, 10, and 15 Hz was 37.62±4.13, 103.89±33.15, and 155.95±30.16 beats per minute before 100 ng/kg paraoxon administration, 47.78±22.82, 132.94±12.43, and 218.33±9.62 beats per minute before 100 µg/kg paraoxon administration, and 41.67±4.41, 85.00±7.64, and 203.33±6.67 beats per minute before 1.0 mg/kg parathion administration. (B) The higher dose of paraoxon (100 µg/kg) slightly but not significantly potentiated ACh-induced bradycardia. Data are presented as mean ± SE; n = 4–7 guinea pigs.

Figure 3. Paraoxon potentiates electrical field stimulation (EFS)-induced contractions in isolated guinea pig trachea and ileum.

Contractions in response to EFS (10 Hz, 100 V, 0.2 ms, 5 s duration every 30 s) and ACh (5 µM) were measured in trachea (A) and ileum (B) before and after addition of paraoxon or vehicle (DMSO, 0.1% final). EFS induced contraction was measured for 30 min after drug treatment, while ACh-induced contractions were measured 35 min after drug treatment. (A) Paraoxon potentiated EFS-induced contractions in the trachea with significant effects at 360 nM; paraoxon at 100 and 360 nM significantly potentiated ACh-induced contractions. (B) In contrast, paraoxon did not significantly potentiate either EFS-induced or ACh-induced contractions in the ileum even at concentrations that significantly inhibited AChE activity. (C) Concentrations of physostigmine that significantly inhibited AChE activity to the same degree as paraoxon did not potentiate EFS-induced contractions in the ileum. Data are presented as mean ± SE; n = 4–6 guinea pigs (*p<0.05).

Figure 4. Neuronal M2 muscarinic receptor mRNA expression is not altered by paraoxon.

(A) M2 receptor transcript levels in human SN-N-SH cells were not changed by exposure to paraoxon (0.1–1000 nM) for either 4 hr (gray bars) or 24 hr (black bars), while TNFα (2 ng/ml, 4 hour exposure, white bar) significantly suppressed M2 expression. (B) Exposure for 24 hr to a similar range of paraoxon concentrations had no effect on M2 mRNA levels in rat sympathetic neurons. Data are expressed as a % of control levels (cultures exposed to 0.1% DMSO) and are presented as mean ± SE; n = 3 independent cultures per treatment group (*p<0.05).

Figure 5. Paraoxon does not alter expression of M2 muscarinic receptor protein.

Rat sympathetic neurons in cell culture (A) or Cos-7 cells transfected with full length cDNA encoding human M2 receptor (B) were treated with paraoxon (1–1000 nM) or vehicle (0.1% DMSO) for 24 hr in the absence or presence of carbachol (1 mM). Muscarinic receptor expression was determined as specific binding of [³H]-NMS (1 nM; surface receptors) or [³H]-QNB (1 nM; total receptors) in the absence (total) or presence (non-specific binding) of atropine (0.1 M). In both cell types, carbachol significantly decreased [³H]-NMS (A and B, open bars), but not [³H]-QNB (C, open bars) binding. In both sympathetic neurons (A) and Cos-7 cells (B), paraoxon had no effect on [³H]-NMS or [³H]-QNB binding in the absence or presence of carbachol. Data are represented as mean ± SE; n = 3–5.

Figure 6. The organophosphorus fluorophosphonate (FP) probe does not phosphorylate M2 muscarinic receptors in guinea pig heart.

Guinea pig heart cell membrane preparations with abundant expression of M2 muscarinic receptors or BSA were reacted with a fluorophosphonate tethered to a biotin group (FP-biotin) for 24 hr prior to separation by acrylamide gel electrophoresis. Blots of these gels were probed with streptavidin tagged with an infrared fluorophore (A, red in the merged image in panel C) to localize the biotin tag and with anti-M2 receptor antibody conjugated to a different infrared fluorophore (B, green in the merged image in panel C). As indicated in the merged image (C), proteins in the heart membranes that were biotinylated by the FP probe (red) did not co-localize with bands recognized by the anti-M2 receptor antibodies (green).