Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice

Abstract

Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic neurotransmission. Overstimulation of cholinergic receptors by excess acetylcholine is known to be lethal. However, AChE knockout mice live to adulthood, although they have weak muscles, do not eat solid food, and die early from seizures. We wanted to know what compensatory factors allowed these mice to survive. We had previously shown that their butyrylcholinesterase activity was normal and had not increased. In this report, we tested the hypothesis that AChE−/− mice adapted to the absence of AChE by downregulating cholinergic receptors. Receptor downregulation is expected to reduce sensitivity to agonists and to increase sensitivity to antagonists. Physiological response to the muscarinic agonists, oxotremorine (OXO) and pilocarpine, showed that AChE−/− mice were resistant to OXO-induced hypothermia, tremor, salivation, and analgesia, and to pilocarpine-induced seizures. AChE+/− mice had an intermediate response. The muscarinic receptor binding sites measured with [³H]quinuclinyl benzilate, as well as the protein levels of M1, M2, and M4 receptors measured with specific antibodies on Western blots, were reduced to be approximately 50% in AChE−/− brain. However, mRNA levels for muscarinic receptors were unchanged. These results indicate that one adaptation to the absence of AChE is downregulation of muscarinic receptors, thus reducing response to cholinergic stimulation.

Introduction

Acetylcholinesterase (AChE; EC 3.1.1.7) has a crucial role in cholinergic neurotransmission, hydrolyzing the neurotransmitter acetylcholine to terminate nerve impulse transmission. Acute AChE inhibition by nerve agents or organophosphorus pesticides may be lethal. No case of total AChE deficiency in the human population has ever been reported, leading to the speculation that inactive AChE mutations may be embryonically lethal. Therefore, it was a surprise to find out that AChE knockout mice without AChE activity survived to adulthood Duysen et al., 2002, Li et al., 2000, Xie et al., 2000.

The absence of AChE activity most likely results in abnormally high levels of acetylcholine in the cholinergic synapses. AChE−/− mice have motor tremor and pinpoint pupils, signs indicative of the presence of excess acetylcholine. Although acute overstimulation of acetylcholine receptors by excess acetylcholine is known to be lethal (Mileson et al., 1998), surprisingly, AChE−/− mice live to adulthood when maintained on a liquid diet (Duysen et al., 2002) and many have survived for nearly 2 years. Death occurs from seizures. The cholinergic marker, choline acetyl transferase, showed normal anatomical distribution in AChE−/− brain, indicating that the absence of AChE does not affect the development of cholinergic pathways in the central nervous system (Mesulam et al., 2002).

The mice live despite the complete absence of AChE, suggesting that they have adapted to the absence of AChE, or that a backup enzyme substitutes for the missing AChE activity. Butyrylcholinesterase (BChE; EC 3.1.1.8) did not undergo compensatory increases in the AChE−/− mouse (Li et al., 2000). However, inhibition of BChE was lethal to AChE−/− mice (Xie et al., 2000). It is possible that the normal level of BChE plays a role in keeping AChE−/− mice alive by hydrolyzing acetylcholine Li et al., 2000, Mesulam et al., 2002. The present study examined whether AChE knockout mice have adapted to the absence of AChE by downregulating muscarinic receptors. Several other adaptation mechanisms are possible, but have not yet been tested. For example, nicotinic receptors may be downregulated, and there may be changes in rates of acetylcholine synthesis and release.

The action of acetylcholine is mediated by nicotinic and muscarinic acetylcholine receptors (mAChRs). The mAChR family belongs to the G protein-coupled receptor gene superfamily and consists of five subtypes (M1–M5), which regulate numerous fundamental physiological processes, including motor control, temperature regulation, pain perception, learning, and memory Messer et al., 1990, van der Zee and Luiten, 1999. In the peripheral nervous system, mAChR mediates smooth muscle contraction, glandular secretion, and cardiac function Caulfield and Birdsall, 1998, Eglen, 2001. Acetylcholine is known to regulate the level of both nicotinic and muscarinic receptors in many systems. Chronic agonist stimulation of mAChR results in desensitization and downregulation of mAChR (Honda et al., 1995). Downregulation of muscarinic receptors is generally accompanied by decreased sensitivity to muscarinic agonists and increased sensitivity to muscarinic antagonists. Conversely, upregulation of mAChR is found after chronic administration of muscarinic antagonists (Ben-Barak and Dudai, 1980). Therefore, we tested whether mice without AChE have adapted to the absence of AChE by downregulation of cholinergic receptors. We found that AChE knockout mice have a significant reduction in the number and responsiveness of muscarinic receptors, indicating that the chronic absence of AChE produces marked changes in the cholinergic system. Downregulation of muscarinic receptors is one of the mechanisms that explains the survival of AChE−/− mice.