Fig. 11.1 On the top of the Þgure is acetylcholine, which is the classical neurotransmitter released from the small clear vesicles of the post-ganglionic parasympathetic nerve terminals. Below it is vasoactive intestinal peptide (VIP), which is a non-cholinergic, non-adrenergic (NANC), non-conventional peptide transmitter released from the large dense-core vesicles of the post-ganglionic parasympathetic nerve terminals. Their release occurs concomitantly, which leads to a coupling between (1) the rapidly occurring acetylcholine-induced ion channel opening and production of watery secrete during the stimulation phase and (2) a prolonged VIP-induced, gene transcription-mediated restoration during the recovery phase. This Þgure shows the Þrst isoform of the prepropolypeptide, which is bioprocessed into a signal peptide (1Ð20), propeptide (21Ð79), intestinal peptide PHV-42 (81Ð122), intestinal peptide PHM-27 (81Ð107), vasoactive intestinal peptide (VIP) (125Ð152, darker shading in the Þgure), and a propeptide (156Ð170). In the other slightly different pre-propolypeptide precursor the same mature VIP peptide forms the residues 124Ð151

parasympathetic nervous system the exocrine gland function can be divided into two distinct phases. During the resting phase the acinar cell is loaded with Cl−and K+. During the stimulation phase acetylcholine leads to a discharge of the osmotic potential by opening two ion channels, which leads to secrete ßow and helps to maintain the prolonged secretory phase of the acinar cell (Fig. 11.3). To this ßuid the sympathetic nervous system, by its direct action on acinar cells, adds a protein-rich and mucin-rich viscous component. Interestingly, anti-cholinergic medication

is today among list of exclusions in the diagnostic criteria and, vice versa, cholinergic agonists, sialogogues, pilocarpine, and cevimeline are used to stimulate salivary ßow.

Two to three myoepithelial cells surround individual acini. Naturally also their function is divided into two distinct phases. During the resting phase they provide dynamic structural support for the underlying acini, which become swollen due to Þlling with mucin hydrogel or secretory protein-rich granules. In sialosis in diabetic, anorectic, or alcoholic patients the volume of individual cells and the size of the whole gland increase. Filling phases are extended, because the acinar cells are not properly and regularly emptied due to an autonomic neuropathy, lack of stimuli, or ÒliquidÓ alcohol diet. Upon autonomic nervous system stimulation in healthy individuals myoepithelial cells receive a dual stimulus from sympathetic and parasympathetic branches, which acts synergistically to promote outßow of saliva and salivary mucins. This phenomenon, a short-term improvement of salivary ßow, has been also observed in patients with pSS [12].

In addition to its function as a secretagogue, acetylcholine and its α7-nicotinic acetylcholine receptor form the central component in Òthe cholinergic anti-inßammatory pathwayÓ [13].

11.3Neuropeptides

11.3.1 Acinotrophic Neurogenic Stimuli

Although the two phases of the sympathetic, parasympathetic, vascular, acinar, and myoepithelial cell function are often referred to as resting and stimulated phases, probably because it is commonplace to refer to measurement of resting (particularly from the minor and submandibular glands) and stimulated (particularly from the major salivary glands) ßow, ÒrestÓ is by no means total rest for the main player of this review and this enigmatic syndrome, the acinar cell. As a matter of fact, right after the reßex secretory ßow has stopped, follows a very feverous period in the life cycle of the secretory acinar cell because it has to recover from stimulated emptying and

Fig. 11.2 Acetylcholine (black circles) released from the post-ganglionic parasympathetic nerve (PGPN) terminals binds to post-synaptic muscarinic (M3 and M1) receptor. It is via a G-protein coupled to activation of phospholipase C (PLC), which has just cleaved phosphatidyl inositol bisphosphate (PIP2) in the cell membrane to cell membrane-bound diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which has been released into the cytoplasm. IP3 binds to its receptors in the endoplasmic reticulum, which leads to rapid cytoplasmic calcium increase (or coupled with re-uptake, to oscillations) via

Fig. 11.3 During the resting phase Na+/K+/2Cl− co-transporter and Na+/K+-ATPase load (charge) the acinar cell with potassium and chloride. As a result of acetylcholineÐmuscarinic receptor interaction, the acinar cell is stimulated and during stimulation phase it discharges its osmotic potential via opening of the apical Cl− channel allowing chloride to ßow into the acinar

release of Ca2+ through these IP3 receptors. This triggers Ca2+ inßux from the extracellular space along its 10,000fold concentration gradient through store-operated Ca++ SOC channels. Intracellular Ca2+ together with DAG activates in the Þgure the conventional protein kinase C (PKC) molecule into its active counterpart (marked with ). Increase in intracellular Ca2+ activates two ion channels, which lead to production of primary secrete. Notice that at the same time when acetylcholine is released, also VIP (white triangles) is concomitantly released

lumen. Sodium follows due to the electrical charge and water due to the osmotic gradient. Opening of the basolateral K+ channel and secretion of K+ into the extracellular space lead to improved function of the Na+/K+/2Cl− co-transporter, which stimulates Na+/K+-ATPase so maintaining and increasing the effective secretory process

eventual cellular damage inßicted by forceful secretory activity burst, to be prepared for the next activity cycle. Because reßectory ßow is stimulated by post-ganglionic parasympathetic nerve Þbers, it would be reasonable to expect that

there is a coupling between the extent of stimulation (secretory work) of the acinar cells and their subsequent recovery, which is mediated by some neuronal stimulus released concomitantly with acetylcholine.

type of transmitter in its synapses, e.g., the post-ganglionic parasympathetic nerve terminals release acetylcholine (Figs. 11.1 and 11.2). This view has long been challenged by discovery of neuropeptides, excitatory and inhibitory amino acids, purines, nitric oxide, carbon oxide, and other Ònon-adrenergic, non-cholinergicÓ (NANC) transmitters in nerve cells and terminals containing conventional neurotransmitters. In exocrine gland function vasoactive intestinal peptide (VIP), released from the very same post-ganglionic parasympathetic nerve terminals which release acetylcholine, raises particular interest (Fig. 11.2) [14, 15, 16].

The importance of the nervous system for the exocrine glands is illustrated by the effect of surgical, chemical, or functional parasympathectomy, the last mentioned being accomplished by feeding rats with only liquid food instead of solid pellets, which in the long term leads to glandular disuse atrophy. Similar atrophy was not induced upon prolonged treatment with antimuscarinic agents and parasympathetic muscarin receptor agonists did not prevent it, so it is hardly due to acetylcholine deprivation. In contrast, if VIP was administered, the atrophic effect of parasympathectomy was prevented and acinar cells become enlarged, indicating an acinotrophic effect for VIP.

Physiological local VIP release as a result of stimulation of the post-ganglionic parasympathetic nerves may help to maintain acinar cells and promote their recovery [17]. These basic neurophysiological experiments suggest that acinotrophic neurogenic stimuli are released at the same time when also acetylcholine is released, during neuronal stimulation of the glands. They are released in the immediate vicinity of the putative acinar target cells and in proportion to the parasympathetic stimulation. Thus, the multiplicity of neurotransmitters in the postganglionic neurons could reßect their complex function, coupling functional stimulation of the secretory target cells with the long-term maintenance of these very same cells (the proportional response principle). The problem with this type

of coupling of acetylcholine and NANCs release is that it can lead to inßammation and depletion.

11.3.2 Neurogenic Inflammation

Inßammation is often in clinical medicine seen as a pathological condition, which needs to be symptomatically treated, in increasing order of potency, with non-steroidal, steroidal, or biological anti-inßammatory drugs. If inßammation is immunologically driven, immunomodulatory or disease-modifying drugs are used. In contrast, in wound healing inßammation is envisioned as an early and necessary phase of subsequent repair. Necrotic tissues need to be phagocytosed and degraded and microbes killed, blood ßow needs to increase to improve oxygenation and provision of nutrients, which are in increased demand, and local cytokines must be produced to orchestrate the complicated healing cascades. It is therefore, perhaps, not so surprising that neuropeptides with trophic effects, supporting repair, also have pro(and anti-)inßammatory actions; those dealt with in this chapter are shown in Table 11.1. Neurogenic inßammation was Þrst realized as a wheal and ßare response evoked by an axon reßex, but understanding of the sophistication of this potential has since increased tremendously. Neuropeptides can be considered as extravascular, intra-axonal potent Òneuronal cytokines,Ó which are delivered close to the effector cells via a special mechanism of delivery, release from the nerve terminal.

Apart from the molecular coupling of the nervous system and inßammation with each other, the nervous and the immune systems are anatomically coupled through the autonomic nervous system [47]. There are rich autonomic neural connections with lymphoid tissue, including thymus, bone marrow, lymph nodes, spleen, and gut-associated lymphatic tissue [71, 72, 26].

Substance P is a short, 11-amino acid long neuropeptide belonging to the tachykinin family [73], widely distributed throughout the nervous system, including salivary glands [74]. Neuropeptides are synthesized through gene transcription and translation, not enzymatically, and

thus differently from the classic neurotransmitters [29]. Preproneuropeptide synthesis occurs in the neuronal cell body far away from the site of neuropeptide storage and release, the nerve terminal, or site of action [75]. From the perinuclear region the newly synthesized neuropeptide precursors are transported (and simultaneously processed, maturation) in vesicles in the extravascular, intra-axonal space along microtubules to the nerve terminals [76]. If coupling of the synthesis and fast intra-axonal transport (5Ð40 cm/day) or ÒdeliveryÓ does not meet neuropeptide demand, neuropeptide depletion follows. This happens rarely with enzymatically and locally synthesized classical neurotransmitters, which can be recycled and form a much easier replenishable transmitter pool. This has some potentially important consequences [32].

In peripheral tissues substance P is released from unmyelinated polymodal nociceptors. Apart from reßexes substance P release can be directly induced by molecules, like capsaicin from paprika. Substance P acts via endogenous neurokinin (NK) 1 receptor [77, 78, 79]. The NK1 receptor is widely distributed in the central and peripheral nervous systems [80, 81] and in nonneuronal structures [82, 31]. High levels of mRNA for NK1 are expressed in the salivary glands of rats [83]. Substance P up-regulates aromatase [84]. Although it is not very likely, substance P might play a role in focal adenitis or substance P depletion might protect from excessive host responses [82, 28, 31].

Calcitonin gene-related peptide (CGRP) is mainly produced in the nervous tissue and its receptor CALCR is expressed throughout the body [46]. CGRP is the most potent endogenous neurogenic vasodilator and a likely participant in the migraine development [48]. Although many earlier papers suggested a role for CGRP in neurogenic inßammation, more recent studies suggest that CGRP does not induce neurogenic inßammation in humans or rodents but only causes a neurogenic vasodilatation by acting via CALCR and cAMP on vascular smooth muscles [85]. It is not known if this potent vasodilator plays a role in the SS-related vascular phenomena, like the various phases of the RaynaudÕs phenomenon or the regulation of the precapillary

sphincters of the metarterioles, which forms as an essential component in the regulation of salivary ßow at rest and upon stimulation.

Neuropeptide Y (NPY) is in the central nervous system secreted by the hypothalamus. NPY receptors (Y1RÐY5R) are widely present in many different cells and tissues [86]. NPY is ubiquitous in sympathetic nervous terminals, including those of resistance blood vessels [87]. As a matter of fact, one of the Þrst effects of NPY reported in the literature was its sympathetic vasoconstrictor effect resistant to α-adrenoceptor blockade [88], which although not strong by itself alone, greatly enhanced the effects of noradrenaline released from the same post-ganglionic sympathetic nerve terminals [89]. It is unknown if NPY perhaps directly mediates vasoconstriction or indirectly potentiates the vasoconstrictor effects of noradrenaline in the precapillary sphincters of the metarterioles. It could turn them ÒoffÓ (or in case of ceased release turn them ÒonÓ). NPY could also participate in the regulation of the myoepithelial cells.

NPY is involved in the innervation of lymphatic organs and in immune cell functions. NPY receptors are expressed by leukocytes [90]. NPY mediates proliferation, adhesion, and differentiation of T lymphocytes toward Th2 cells, but vice versa, in a typical bidirectional communication also many immune-inßammatory molecules regulate NPY synthesis and/or release [91]. NPY stimulates leukocyte trafÞcking and B lymphocytes, including their immunoglobulin production [59], but it is not known if these effects play a role for the autoantibody production in SS.

Perhaps the most interesting neuropeptide from the SS point of view is vasoactive intestinal peptide (VIP), due to its potential as an acinotrophic factor. It might play a role for the autoimmune aspects of the syndrome. Acinar cell, which is not properly maintained, undergoes apoptosis or even necrosis and its molecular components can be abnormally degraded. This could lead to a local release, processing, and presentation of hidden or ÒunforeseenÓ epitopes (or actually endotopes) instead of the dominant epitopes, which are produced under Òbusiness as usualÓ type of apoptosis and against which the host therefore has developed immunologic