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The neurotransmitter acetylcholine (ACh) is involved in memory and cognitive functions, which normally decline with age. In this chapter, we describe qRT-PCR and immunohistochemical protocols for measurement of muscarinic ACh receptor M1 (m1AChR) levels in the brains of middle-aged rats, with and without administration of grape seed proanthocyanidin extract (GSPE) and exercise training. The analyses revealed that the interventions led to an increase in m1AChR mRNA and protein levels in the CA1 subfield of hippocampus. This would be expected to enhance Ach levels at synapses and thereby boost cognitive ability. The protocols can be applied to m1AChR measurements in neurodegenerative diseases and dementia.

The story of receptors is bound with the history of the recognition of chemical transmission at the periphery and in the central nervous system (CNS). In turn, this recognition is based on the progress in the description of the anatomy of the peripheral and central nervous systems. This progress was described in detail several times (see, for example, Brazier, 1959; Karczmar, 1967, 1986; Pick, 1970); at this time, it suffi ces to list the main steps of the early history of this progress and the principal authors who were involved (Table 4-1). As shown in the table, by the 18th century the main characteristics of the central, autonomic, and peripheral motor systems were laid down; in fact, even 250 years earlier the great da Vinci had rendered in his fi gures with much precision and few errors the details of the nervous systems (and other tissues).

Muscarinic acetylcholine (ACh) receptors (mAChRs) are members of the 7 transmembranespanning, guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily. There are 5 subtypes of mAChRs, and these mediate the majority of the actions of ACh in both the central nervous system (CNS) and the periphery. For example, peripheral mAChR activation results in a reduction in heart rate and vasodilatation of vascular beds, increases in exocrine secretions from sweat and lacrimal glands, and contraction of smooth muscle in the gastrointestinal tract, airways, ciliary body, and iris sphincter. All 5 subtypes of mAChRs are expressed in the CNS and are postulated to play a role in learning and memory, arousal, REM sleep, psychotic states, control of movement, thermoregulation, reward behaviors, and the generation of epileptic foci. This chapter provides an overview of current knowledge regarding the structure, function, and pharmacology of the mAChRs, with a particular emphasis on their roles in the CNS. For a historical introduction, see Chapter 4 A-E.

Endothelin-1 (ET-1) and the muscarinic agonist carbachol do not affect the basal force of contraction, but induce a pronounced negative inotropic effect in the presence of β-adrenoceptor stimulation in the canine ventricular myocardium. We studied the influence of the phosphatase inhibitor cantharidin on the negative inotropic effect of ET-1 and carbachol in isolated canine right ventricular trabeculae and single ventricular myocytes. In the presence of 100 nM norepinephrine (NE), 10nM ET-1 and 30 nM carbachol induced a negative inotropic effect of an identical extent. Cantharidin at 10 μM did not affect the basal force of contraction and the positive inotropic effect of NE mediated by β-adrenoceptor stimulation. Cantharidin (10 μM) markedly attenuated the negative inotropic effect of ET-1, but it did not affect the negative inotropic effect of carbachol. At 30 μM, cantharidin induced a positive inotropic effect and enhanced the positive inotropic effect of NE. Cantharidin (30 μM) suppressed significantly the negative inotropic effect of carbachol (30 nM and 100 nM). In canine single ventricular myocytes, ET-1 (10nM) or carbachol (30nM) did not affect the baseline level of cell shortening and the amplitude of intracellular Ca2+ transients, while they inhibited the NE (30 μM)-induced increases in cell shortening and Ca2+ transients. Cantharidin (10 μM) attenuated the inhibitory action of ET-1, but did not affect the effects of carbachol in the presence of NE. These results indicate that the activation of phosphatase that is susceptible to cantharidin is involved in the negative inotropic effect of both ET-1 and carbachol. The extent of contribution of phosphatase activation appears to be greater in the ET-1-induced negative inotropic effect than in the effect of the muscarinic receptor agonist carbachol in the canine ventricular myocardium.

It is generally accepted that neurotransmitters are involved in hypoxic chemotransmission of the carotid body. The release of neurotransmitters from the glomus cell, a putative chemoreceptor cell, appears to be triggered by an influx of calcium and subsequent increase in intracellular calcium ([Ca2+]i). Several reports indicate that L-type and some other types of voltage-gated calcium channels are responsible for neurotranmitter release from glomus cells. Since these channels are activated by depolarization of the cell membrane, mechanisms of hypoxic depolarization of glomus cells has been a major focus of investigation (Shirahata and Sham, 1999).

Decades of studies have shown the presence of neurotransmitters in the glomus cell, the putative chemosensory cell, and the involvement of neurotransmitters in carotid body chemotransmission (Gonzalez et al, 1994). We have been proposing that ACh is a major excitatory neurotransmitter in the cat carotid body (Fitzgerald, 2000). ACh fulfills most of criteria as an excitatory neurotransmitter in the carotid body. Regarding the presence of cholinergic receptors, our immunohistological study has shown that α7 subunits of nicotinic ACh receptors (nAChRs) locate at chemoreceptor nerve endings (Shirahata et al, 1998), and that α4 and β2 subunits of nAChRs in glomus cells (Ishizawa et al, 1994). Expression or localization of other subunits of nAChRs or subtypes of muscarinic ACh receptors (mAChRs) has not yet been known. Neuronal nAChRs are ligand-gated cation channels. They are composed of two a (a2-6) and three β (β2-4) subunits, or of five a (a7-9) subunits. Among variable combinations, α3β4, α4β2, and a7 types appear to be the major nAChRs, and they are widely but distinctively expressed in the nervous system (Lukas et al, 1999). Muscarinic AChRs are pharmacologically and molecular biologically classified into 5 subtypes (M1-M5). They are distributed in the nervous system, smooth muscles and gland (Caulfield and Birdsall, 1998). The purpose of the current study is to investigate the gene expression of major cholinergic receptors and to localize the receptor proteins in the cat chemosensitive unit (the carotid body and the petrosal ganglion).

Peripheral arterial chemoreceptors are essential to respiratory and cardiac homeostasis. Although the components of these chemoreceptors lie within a tiny structure, the carotid body, the physiological affects resulting from activation of the peripheral arterial chemoreceptors have profound effects on arousal responses and cardiorespiratory responses to hypoxia and asphyxia (Gonzalez et αl., 1994). In response to hypoxia, hypercapnia and acidosis the glomus cell within the arterial chemoreceptor depolarizes; intracellular calcium levels rises, and neurotransmitters are released. These neurotransmitters bind to autoreceptors on the glomus cell and postsynaptic receptors on the carotid sinus nerve. Binding of neurotransmitters to autoreceptors on glomus cells regulates further neurotransmitter release, while binding to postsynaptic receptors on chemoafferent nerve fibers results in electrical output through the carotid sinus nerve (for reviews, see Gonzalezet al., 1994; Prabhakar 1994).

The most fundamentally necessary substrate for life is oxygen.The human organism cannot be without it for more than a few minutes without doing irreversible damage to neural structures. This substrate is used to oxidize glucose, which process provides the organism with the energy required for all of life’s processes. Well-appreciated is the fact that the carotid body is the principal detector of systemic hypoxia. The aortic bodies also play a role, but appear to be in humans at least considerably less important. We have learned in this conference that the carotid body also increases its neural output to the nucleus tractus solitarius in response to reduced glucose in blood or perfusion fluids.lt is therefore somewhat amusing as well as frustrating that even though 250 years have passed since the first report of the carotid body in 1743, the precise steps describing how this all-important structure sends its message to the nucleus tractus solitarius continue to remain mysterious.Generations of investigators have tried to determine these mechanisms. And though considerable progress has been made, no definitive answer can yet be provided.interrelationship in the carotid body. Bairam and her colleagues (2000) using a different preparation also found that cholinergic agonists delivered exogenously controlled the release of catecholamines. That cholinergic mechanisms are found to control catecholaminergic release in the carotid body is not altogether surprising. The autonomic nervous system controls the heart’s stroke volume and rate with a balance and interaction of the sympathetic and parasympathetic nervous systems on the heart and on each other. In the central nervous system there are several instances of muscarinic and nicotinic modulation of dopamine and noradrenaline release (Clarke et al., 1996; DeKlippel et al., 1993; P. Izurieta-Sanchez et al, 2000; Smolders et al., 1997). One study suggested a potential source for the difference between release patterns for DA and NE. Presynaptic nicotinic receptors associated with striatal DA terminals differed pharmacologically from those on hippocampal NE terminals. Nigrostriatal DA neurons expressed mainly a4, a5, and 02 nicotinic receptor subunits, while hippocampal NE neurons expressed a3, p2, and P4 subunits. This situation is consistent with the reports of several other glomus cell heterogeneities. It seems entirely possible that not all glomus cells have exactly the same complement of neurotransmitters nor the same set of nicotinic receptors. Further study of neurotransmitter interrelationships will undoubtedly help to bring greater clarity to these interactions so fundamental to the chemotransduction of hypoxia, and perhaps reveal clues as to the mechanisms involved in the transduction of hypoglycemia.

Neurotransmitters are likely to play a key role for hypoxic chemotransmission in the carotid body. Among several neurotransmitters, ACh appears to be a major excitatory neurotransmitter in the cat carotid body (Fitzgerald, 2000). ACh may act directly on the afferent nerve endings and generates action potentials (Hayashida et al., 1980; Zhong & Nurse, 1997). Alternatively, ACh may work through nicotinic ACh receptors (nAChRs) in the carotid body chemosensory cells, glomus cells, and modulate the release of neurotransmitters (Fidone et al., 1990). Our recent data have shown the presence of ¦A3, ¦A4, ¦Â2 and ¦Â4, but not ¦A7, subunits of nAChRs in the cat glomus cells (Hirasawa et al., in this book; Shirahata et al., 1998). Nicotinic AChRs belong to ligand-gated ion channels, that are composed of five receptor subunits. Their subunit compositions categorize nAChRs into three receptor types: (1) muscle type nAChRs, (2) heteromeric type neuronal nAChRs that consist of ¦A subunits (¦A2-6) and ¦Â subunits (¦Â2-4), and (3) homomeric type neuronal nAChRs that consist of only a subunits (¦A7-9). Their stoichiometry and their electrophysiological characteristics depend on the subunit compositions (McGehee & Role, 1995; Lindstrom et al., 1996). Since neuronal nAChR channels are permeable to Ca2+ and Na+ ions, their activation could significantly influence the excitability and fonction of glomus cells. In this study we examined electrophysiological and pharmacological characteristics of nAChRs in glomus cells and infer their role in the excitability of glomus cells.

Sjogren’s syndrome (SS) is characterized by symptoms of dryness of the eyes and mouth associated with lymphoid infiltrates of the lacrimal and salivary glands. Confusion exists about the criteria for diagnosis of this disease, 1 but the frequency, according to a newly proposed international criterion that requires either a characteristic minor salivary gland biopsy or the presence of an antibody against SS-A/SS-B antigen, 2 is about 0.5% of adult women. SS may exist with other autoimmune disorders (secondary SS associated with rheumatoid arthritis, systemic lupus erythematosus or scleroderma), or as a primary condition. Primary and secondary SS are systemic autoimmune disorders, although the most recognizable manifestations are the ocular and oral components of the disease.

Previous pharmacological, immunocytochemical, electrophysiological, and microfluorometric studies have suggested that acetylcholine (ACh) is a critically important excitatory transmitter in the chemotransduction of hypoxia by the cat carotid body (CB). With the use of HPLC this study shows that the in vitro cat CB releases ACh under normoxic conditions; this release is increased when the CB is challenged with hypoxia. The preliminary observation that greater amounts of ACh are liberated in the presence of gallamine and AFDX116 suggests the presence of functioning M2 muscarinic receptors on the glomus cells of the CB.

M1 muscarinic receptors (M 1 mAChR) have an important role in cognitive processing relevant to Alzheimer’s disease brains (AD)1–5. M1 mAChR are relatively unchanged in AD1–5 and therefore may serve as a target for an anti-dementia drug treatment. However, some of the tested muscarinic agonists were not highly M1 selective, had major clinical limitations and showed disappointing clinical results in AD 4,6,7. Thus the proof of clinical concept could not be shown.

Many frontiers in the modeling of the ventilatory system and its control are at the cellular and molecular levels. The carotid body’s response to hypoxia is no exception to this. A commonly accepted model of the carotid body’s chemotransduction of hypoxia presents the transmitter-laden glomus cell being depolarized by the hypoxic challenge in as-yet-to-be­determined processes. This depolarization activates voltage-gated calcium channels with the subsequent entrance of extracellular calcium. This event, in turn, precipitates the release of several transmitters. Many investigators have reported the hypoxia-generated release of catecholamines from the carotid body. And recently we have reported the release of acetylcholine (ACh) from the cat carotid body during hypoxia1. These transmitters are presumed to bind to “postsynaptic” receptors located on the apposed sensory afferent fibers which have their cell bodies in the petrosal ganglion with their axons terminating in nucleus tractus solitarii. The transmitters are also presumed to bind to “presynaptic” autoreceptors on the glomus cells. Over the last decade our working hypothesis has been that ACh is the (or at least one) critically important excitatory transmitter. And in the mid 1990s we generated some neurophysiological recordings2 which suggested the presence of both M1 and M2 muscarinic receptors in the carotid body. The data could not answer whether the receptors were located postsynaptically, presynaptically, or both. Since then we have reported preliminary results suggesting that they are at least located on the glomus cell.

SNI-2011, (±)-cis-2-methylspiro(1,3-oxathiolane-5, 3’-quinuclidine) hydrochloride hemihydrate, is a structurally rigid analog of acetylcholine (ACh) that has been described as for muscarinic M1 (M1) receptors selective agonist (Fisher et al., 1991; Ono et al., 1988;Iga et al., 1996). SNI-2011 was shown to be a selective agonist for M1and M3 receptors (Hargreaves et al., 1992). The intravenous administration of SNI-2011 induces a long-lasting salivation in rats and dogs (Iwabuchi and Masuhara, 1994; Masunaga et al., 1997).

A dataset constisting of 91 M1, M2 and M3 muscarinic antagonist1–3 was considered in order to create a 3D-QSAR model able both to predict the activities of compounds not yet synthesized and to evidentiate the structural features of the ligands for a specific receptor subtype selectivity. For example, M2 selective antagonist could be very useful in the treatment of Alzheimer disease, a pathological state in which central cholinergic activity is reduced. In fact, a possible approach to increase the central cholinergie tone could be the blockade of the presinaptic autoreceptors, that modulate Ach release and probably belong to the M2 subtype.

Pharmacological treatment of schizophrenia has been traditionally dominated by dopamine D2 receptor antagonists, known to cause severe extrapyramidal side effects, which can be attributed to the blockade of D2 receptors in the striatum. Current antipsychotic research focuses on compounds with multireceptorial activity (different dopamine receptor subtypes, serotonine and adrenerg and muscarinic receptors has been sudied). Recent observations indicate, that control of both dopaminergic and serotoninergic systems is important for adequate antipsychotic therapy. It has been reported, that 5-HT1A receptor agonists reverse antipsychotic induced catalepsy.