Enhancement of maximal activation of neuronal nitric oxide synthase at muscarinic M₁ receptors following prolonged agonist treatment

Abstract

It was previously believed that the neuronal type of nitric oxide (NO) synthase was constitutive in nature, and that changes in the concentration of intracellular Ca²⁺ represent the sole input that regulates its activity. Recent reports, however, suggested that this enzyme could also be induced under certain conditions. We report here that prolonged stimulation of M₁ muscarinic acetylcholine receptors results in potentiation of maximal receptor-mediated activation of neuronal NO synthase in Chinese hamster ovary cells. This effect was dependent on the concentration of agonist during the treatment and was abolished by a muscarinic receptor antagonist. These findings are important for understanding the sequelae of prolonged administration of muscarinic agonists in vivo.

Introduction

The effects of nitric oxide (NO) on blood vessels and cGMP formation have long been known before it was discovered that it can be produced endogenously by a group of enzymes called NO synthases (Ignarro et al., 1981; Palmer et al., 1987, Palmer et al., 1988). NO also has diverse effects on neurons, and it has been shown to play an important role in learning (Dinerman et al., 1994), neurotoxicity (Nowicki et al., 1991; Dawson, 1994) and in regulation of neuronal excitability (Bagetta et al., 1992). Three isoforms of NO synthase have been cloned. Two isoforms are constitutive and their activity is Ca²⁺-calmodulin dependent (Bredt and Snyder, 1990). These enzymes are expressed predominantly in neuronal and endothelial cells (Bredt and Snyder, 1992). The third is Ca²⁺ independent and its expression is induced by agents such as gamma-interferon and tumor necrosis factor and by foreign agents such as bacterially-derived lipopolysaccharides (Bredt and Snyder, 1994).

Recent evidence, however, has shown that the activity of neuronal NO synthase is not purely constitutive, since the level of expression of the enzyme is altered under certain conditions. These conditions include brain lesions (Yu, 1994) and ischemia (Kato et al., 1994). Since NO is not stored in neurons, its rate of formation is dependent on the level of activity of neuronal NO synthase, which in turn is dependent on the level of expression of the enzyme. In this report we used Chinese hamster ovary (CHO) cells which co-express M₁ muscarinic receptors and neuronal NO synthase to investigate whether the activity of this important enzyme is modulated by prolonged receptor activation. Our data demonstrate that maximal activation of neuronal NO synthase by M₁ muscarinic receptors is markedly potentiated following long-term exposure to the muscarinic agonist carbachol.