Sally A. Waterman

Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren's syndrome.

OBJECTIVE Sjögrens syndrome (SS) is an autoimmune disorder characterized by dry eyes and mouth (sicca syndrome) and lymphocytic infiltration of the lacrimal and salivary glands. Abnormalities of parasympathetic neurotransmission may contribute to the glandular dysfunction. In this study, we used a functional assay to investigate autoantibody-mediated effects on parasympathetic neurotransmission and smooth muscle contraction. METHODS Serum and purified IgG were obtained from patients with primary and secondary SS and from control subjects. Contraction of isolated bladder strips in response to stimulation of M3-muscarinic receptors by a muscarinic receptor agonist, carbachol, or by endogenous acetylcholine released from postganglionic parasympathetic nerves was measured before and after the addition of patient serum or IgG. RESULTS Sera from 5 of 9 patients with primary SS and from 6 of 6 patients with secondary SS inhibited carbachol-evoked bladder contraction by approximately 50%. Sera from these patients also inhibited the action of neuronally released acetylcholine at M3-muscarinic receptors. Sera from 7 of 8 healthy individuals, from patients with rheumatoid arthritis without sicca symptoms, and from patients with systemic lupus erythematosus had no effect. The anti-muscarinic receptor activity was localized in the IgG fraction, since purified IgG from patients with SS also inhibited agonist- and nerve-evoked contractions. In this preliminary study, the autoantibodies seemed to be associated with the presence of bladder symptoms and other autonomic features. CONCLUSION Autoantibodies that act as antagonists at M3-muscarinic receptors on smooth muscle occur in a subset of patients with primary and secondary SS. Their presence in secondary SS was unexpected and provides new evidence for a common pathogenetic link between primary and secondary SS. These autoantibodies appear to contribute to sicca symptoms and may explain associated features of autonomic dysfunction in some patients.

Up-regulation of M3-Muscarinic Receptors in Labial Salivary Gland Acini in Primary Sjögren's Syndrome

M3-muscarinic receptors (M3R) mediate parasympathetic cholinergic neurotransmission to salivary and lacrimal glands, and autoantibodies to these receptors have been implicated in sicca symptoms and autonomic dysfunction in Sjögrens syndrome. We have investigated the expression of M3R in paraffin-embedded labial salivary glands (LSG) from seven patients with primary Sjögrens syndrome (pSS) and five healthy controls using high-resolution confocal microscopy and an affinity-purified goat polyclonal antibody raised against the COOH-terminal sequence of the human M3R. Immunolocalization of M3R was similar in control and pSS glands, with punctate staining of M3R in the basal membrane of acinar cells and in the luminal and abluminal membrane of myoepithelial cells. Bright, granular M3R staining was also detected in the cytoplasm and membranes of all intercalated and striated ducts, and infiltrating lymphocytes in pSS. All immunoreactivity was specifically blocked by the immunizing peptide. An increase in M3R expression specifically in acini in pSS was demonstrated by a 30% increase in receptor number per cluster and a 68% increase in the number of clusters in the membrane. This up-regulation is consistent with inhibition of parasympathetic neurotransmission, possibly by antagonistic autoantibodies to M3R. The up-regulation, rather than down-regulation, of M3R in acini of pSS LSG can explain the effectiveness of muscarinic agonists in treating sicca symptoms in pSS.

Subcellular distribution of aquaporin 5 in salivary glands in primary Sjögren's syndrome.

Secretions from salivary and lacrimal glands are reduced in patients with primary Sjögrens syndrome (PSS). Since aquaporin 5 is involved in transport of water and is present in salivary and lacrimal glands, this protein was thought to have a major role in the pathogenesis of PSS. We used indirect immunofluorescence and an immunoperoxidase technique to assess expression and subcellular localisation of aquaporin 5 in patients and controls. Our results suggest that the distribution and density of aquaporin 5 in salivary glands does not differ between patients with PSS and those without. Thus, the role of aquaporin 5 in the pathogenesis of PSS needs to be reassessed and alternative pathogenetic mechanisms investigated.

THE ROLE OF ENTERIC INHIBITORY MOTONEURONS IN PERISTALSIS IN THE ISOLATED GUINEA-PIG SMALL INTESTINE

1. Peristalsis is a co‐ordinated motor behaviour in which an anally propagated contraction of the circular muscle propels intraluminal contents. The role of excitatory motoneurons in peristalsis is well established; however the role of enteric inhibitory motoneurons is unknown. 2. A combination of a nitric oxide synthase inhibitor and apamin, which blocks relaxation of the circular muscle of guinea‐pig small intestine mediated by enteric inhibitory motoneurons, was used to investigate the role of inhibitory motoneurons in peristalsis in isolated segments of guinea‐pig small intestine. 3. N omega‐nitro‐L‐arginine methyl ester (L‐NAME, 400 microM) and N omega‐nitro‐L‐arginine (L‐NOArg, 100 microM) significantly reduced the threshold volume required to trigger emptying of the intestine. This effect was reversed by L‐arginine (4 mM) and L‐arginine alone increased the threshold volume for initiation of peristalsis. Sodium nitroprusside (0.1‐10 microM), which generates nitric oxide, also increased the threshold volume. L‐NAME, L‐NOArg, L‐arginine and sodium nitroprusside did not alter the maximal intraluminal pressure generated during emptying. Contraction of the longitudinal muscle during the initial phase of fluid infusion was significantly increased by L‐NAME and L‐NOArg and reduced by sodium nitroprusside (1 nM to 10 microM). 4. Apamin (0.5 microM) did not significantly alter the threshold volume necessary to initiate peristalsis or contraction of the longitudinal muscle. However, the maximal pressure generated when the intestine was emptying was significantly increased. Furthermore, short segments of circular muscle contracted apparently randomly, before peristaltic emptying was triggered. 5. A combination of L‐NAME and apamin completely disrupted peristalsis. Contractions of the circular muscle did not always start at the oral end. Stationary contractions as well as contractions propagating orally and anally were observed. 6. It is concluded that enteric inhibitory motoneurons are crucial for peristalsis to occur. They are important in setting the threshold at which peristaltic emptying is triggered, via nitric oxide. They are essential for the propagation of the circular muscle contraction, via an apamin‐sensitive mechanism of transmission. Contraction of the longitudinal muscle during peristalsis is partly inhibited by a nitric oxide‐mediated mechanism.

Accommodation mediated by enteric inhibitory reflexes in the isolated guinea‐pig small intestine.

1. The aim of the present study was to investigate whether the guinea‐pig small intestine shows accommodation to infused fluid, similarly to other regions of the gastrointestinal tract. Tetrodotoxin, papaverine and transmitter antagonists were used to establish the existence of reflex pathways and the nature of the neurotransmitters involved. 2. Compliance, measured as the change in volume of infused fluid divided by the intraluminal pressure change, was reduced by tetrodotoxin (0.6 microM), indicating that there is an overall neurally mediated relaxation of the circular muscle in response to low rates of distension. Papaverine (10 microM) did not have any significant effect on compliance at the low rates of distension, suggesting that the circular muscle is fully relaxed. 3. At each rate of distension, 400 microM N omega‐nitro‐L‐arginine methyl ester (L‐NAME, a nitric oxide synthase inhibitor) significantly decreased the compliance of the intestinal wall, indicating that the circular muscle was relaxed by a nitric oxide‐mediated mechanism. Apamin (0.5 microM), which blocks a component of inhibitory transmission, did not have a significant effect. 4. In control preparations, the intestinal wall was less compliant when distended by fluid at a fast rate, compared with the lower rates of distension. This was not due to changes in passive components of the intestinal wall or a myogenic response to rapid stretch. 5. When the intestine was distended rapidly, 1 microM hyoscine and 100 microM hexamethonium increased intestinal compliance. However, they had no detectable effect on compliance with low rates of distension.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies Raised Against the Second Extracellular Loop of the Human Muscarinic M3 Receptor Mimic Functional Autoantibodies in Sjögren's Syndrome

Functional antimuscarinic M3 receptor (M3R) autoantibodies have been shown to inhibit cholinergic neurotransmission at the postsynaptic level and appear to mediate parasympathetic dysfunction, including sicca symptoms in Sjögrens syndrome (SS). The precise epitope(s) involved in the inhibition of M3R‐mediated cholinergic neurotransmission has not been defined. In this study, an active immunization approach to raise antibodies with functional activity against the second extracellular loop of the M3R was used and their functional properties were compared with those of human autoantibodies. Peptides corresponding to the second extracellular loop of the M3R were used as immunogens in rabbits, and antisera were tested for inhibition of carbachol‐evoked colon smooth muscle contraction in parallel with immunoglobulin G from a patient with SS. Anti‐M3R antibodies were affinity purified on a peptide representing a dominant functional epitope at the COOH terminus of the second extracellular loop of the M3R and tested for concentration‐dependent inhibition. Experimentally raised anti‐M3R antibodies, like the human autoantibodies, showed concentration‐dependent and noncompetitive inhibition of carbachol‐evoked colon contractions. Inhibitory activity was detected by functional assays at concentrations as low as 3 ng/ml, which was below the threshold of detection of antibody by peptide enzyme‐linked immunosorbent assay. It is concluded that the experimentally raised anti‐M3R antibodies share the functional properties of autoantibodies in patients with SS.

THE ROLE OF ASCENDING EXCITATORY AND DESCENDING INHIBITORY PATHWAYS IN PERISTALSIS IN THE ISOLATED GUINEA-PIG SMALL INTESTINE

1. The effects of experimental manipulations to alter the activation of ascending excitatory and descending inhibitory reflexes on peristalsis were studied in isolated segments of guinea‐pig small intestine. 2. The normal site of initiation of the peristaltic contraction, the oral end, was always shifted to a point just anal to a crush which interrupted enteric neuronal pathways. 3. Shortening the functional length of the intestine by a series of crushes, thus effectively reducing the length of enteric neuronal reflex pathways, led to a progressive increase in the threshold distension for triggering the peristaltic contraction. 4. A sleeve placed around the intestine to prevent it from being distended by fluid led to a shift in the site of initiation of the peristaltic contraction to a point just anal to the sleeve. Furthermore, sleeves placed in the anal half of the intestine were able to stop propagation of the contraction. 5. The effect of these manipulations on peristalsis suggests that ascending excitatory and descending inhibitory enteric pathways, activated by distension, determine the pattern of peristaltic activity. The peristaltic contraction is initiated at the oral end as a result of the summation of ascending excitatory inputs and the relative absence of inputs from descending inhibitory pathways at this point. The magnitude of the distension for triggering this contraction is determined by a balance between ascending excitatory and descending inhibitory inputs to the circular muscle. Propagation of the circular muscle contraction requires the activation of ascending excitatory pathways at each point along the intestine and the sequential inactivation of the descending inhibitory reflex pathways anal to the contraction. The propagation of the circular muscle contraction stops when there is no longer a sufficient distension stimulus ahead.

Selective Down-Regulation of Aquaporin-1 in Salivary Glands in Primary Sjögren's Syndrome

Salivary and lacrimal gland secretions are reduced in primary Sjögren’s syndrome (pSS). Aquaporins (AQPs) are involved in transmembrane water transport, and different isoforms show specific cellular and subcellular distributions in salivary and lacrimal glands. Changes in expression of AQP molecules have therefore been suggested to contribute to the glandular dysfunction in pSS. AQP-5 is present in the apical membrane of acinar cells, where it mediates fluid outflow; however, we have recently shown that its expression is not altered in pSS. We therefore studied whether expression of other isoforms of AQP would be altered in pSS. Using high-resolution confocal microscopy, we determined the distribution of AQP-1 and AQP-3 in labial salivary gland biopsies from 11 patients with pSS and 9 healthy controls. AQP-1 is present in myoepithelial cells surrounding acini, and its expression in these cells was decreased by 38% in pSS glands. By contrast, expression of AQP-1 in endothelial cells of nonfenestrated capillaries was not altered in pSS. AQP-3 was expressed in the basolateral membrane of acinar epithelial cells, and its expression was not altered in disease. We therefore conclude that AQP-1 expression in myoepithelial cells is selectively down-regulated in pSS and that myoepithelial cell dysfunction may play a crucial role in the pathology of this disease.

Neural mechanisms underlying migrating motor complex formation in mouse isolated colon

Little is known about the intrinsic enteric reflex pathways associated with migrating motor complex (MMC) formation. Acetylcholine (ACh) mediates the rapid component of the MMC, however a non‐cholinergic component also exists. The present study investigated the possible role of endogenous tachykinins (TKs) in the formation of colonic MMCs and the relative roles of excitatory and inhibitory pathways. MMCs were recorded from the circular muscle at four sites (proximal, proximal‐mid, mid‐distal and distal) along the mouse colon using force transducers. The tachykinin (NK1 and NK2) receptor antagonists SR‐140 333 (250 nM) and SR‐48 968 (250 nM) reduced the amplitude of MMCs at all recording sites, preferentially abolishing the long duration contraction. Residual MMCs were abolished by the subsequent addition of atropine (1 μM). The neuronal nitric oxide synthase inhibitor, Nωnitro‐L‐arginine (L‐NOARG, 100 μM), increased MMC amplitude in the distal region, whilst reducing the amplitude in the proximal region. In preparations where MMCs did not migrate to the distal colon, addition of L‐NOARG resulted in the formation of MMCs. Subsequent addition of apamin (250 nM) or suramin (100 μM) further increased MMC amplitude in the distal region, whilst suramin increased MMC amplitude in the mid‐distal region. Apamin but not suramin reduced MMC amplitude in the proximal region. Subsequent addition of SR‐140 333 and SR‐48 968 reduced MMC amplitude at all sites. Residual MMCs were abolished by atropine (1 μM). In conclusion, TKs, ACh, nitric oxide (NO) and ATP are involved in the neural mechanisms underlying the formation of MMCs in the mouse colon. Tachykinins mediate the long duration component of the MMC via NK1 and NK2 receptors. Inhibitory pathways may be involved in determining whether MMCs are formed.

Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids

1 A recording method was developed to measure physiological parameters of the preparatory and emptying phases of peristalsis in vitro. This method enabled measurement of: the compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension); longitudinal muscle contraction during the preparatory phase; the threshold volume required to trigger the emptying phase; the maximal ejection pressure and the average power generated during the emptying phase, which reflects the rate at which the intestine performs work. Modulation of these parameters by exogenous and endogenous opioids acting at μ, kappa and δ opioid receptors was investigated. 2 The compliance of the intestinal wall during the preparatory phase was reduced by the μ opioid receptor agonist, [d‐Ala2, N‐methyl‐Phe4, Gly5‐ol] enkephalin (DAMGO) but not by the k agonist, dynorphin, or the δ agonist, [d‐penicillamine2, d‐penicillamine5] enkephalin (DPDPE). Reflex contraction of the longitudinal muscle during the preparatory phase was inhibited by DAMGO, dynorphin and DPDPE. The threshold volume required to trigger the emptying phase of peristalsis was increased by DAMGO, dynorphin and DPDPE. 3 The maximal ejection pressure generated during the emptying phase was reduced by dynorphin and DPDPE, but not by DAMGO. The average power generated by the intestine when emptying was not altered by any of the agonists. 4 Electrically stimulated contractions of longitudinal muscle in strips of longitudinal muscle‐myenteric plexus were not inhibited by DPDPE. Similarly, DPDPE did not significantly inhibit electrically induced contraction of circular muscle in strips of circular muscle‐myenteric plexus. 5 Each of the agonist effects on peristaltic parameters was antagonized by the appropriate antagonist: d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) (μ), norbinaltorphimine (nor‐BNI) (κ), naltrindole (δ). 6 It is concluded that μ and κ agonists act primarily on excitatory circular and longitudinal muscle motor neurones. The δ agonist probably acts on enteric neurones presynaptic to excitatory circular and longitudinal muscle motor neurones. 7 Antagonists for μ, delta and κ receptors did not affect any parameters of peristalsis when the intestine emptied against a low resistance. However, when emptying against a high outflow resistance, the average power generated by the intestine was increased by the κ antagonist, nor‐BNI, but not by CTOP or naltrindole. 8 It is concluded that endogenous opioids appear to have little role in peristalsis when the intestine is working against a low outflow resistance. However endogenous opioids, acting primarily at κ receptors, provide a braking mechanism by inhibiting the emptying phase of peristalsis in conditions in which the intestine empties against a higher resistance.