Joong-Soo Kim

Inhibitory effects of autoantibodies on the muscarinic receptors in Sjögren's syndrome

Sjögrens syndrome (SS) is a systemic autoimmune disease that involves reduced salivary secretions. Recently, circulating autoantibodies from SS patients against the type 3 muscarinic cholinergic receptor (M3R) has been reported in the sera of SS patients. However, the role of these autoantibodies in the development of SS has not been elucidated. In this study, purified IgG was obtained from the sera of 11 SS patients, and its inhibitory effect on the M3R of the salivary glands was evaluated using RT-PCR, microspectrofluorimetry, immunohistochemistry, and Western blot analysis. Stimulation with carbachol (CCh) evoked a [Ca2+]i transient in the fura-2 loaded HSG cells. However, pretreatment of the cells with SS IgG (0.5 mg/ml) for 12 or 24 h significantly reduced the magnitude of the CCh-induced [Ca2+]i transient (CICT). We found that the magnitude of CICT was decreased by 62–45% when cells were pretreated with the SS IgG. However, the [Ca2+]i response to ATP was not altered by the pretreatment of SS IgG. The effect of SS IgG on CICT was abrogated by the inclusion of excessive competitive peptides that encode the amino-acid sequence of M3R, which was not recapitulated by nonspecific peptides. The inhibitory effect of SS IgG on the aquaporin (AQP)-5 expression was also examined. After confirming the apical localization of AQP-5 along with its increase by pilocarpine (10−5 M), we examined whether SS IgG had an effect on pilocarpine-induced AQP-5 trafficking to the apical membrane (APM) using rat parotid acinar cells. After incubating the cells with SS IgG for 12 h, the amount of pilocarpine-induced AQP-5 significantly decreased compared to the control groups. In conclusion, autoantibodies from the SS patients inhibit the function of the human M3R that is mediated by Ca2+ mobilization and AQP-5 trafficking. Our results could partly explain the underlying mechanisms of glandular dysfunction and associated features of impaired autonomic function in SS patients.

Mechanosensitivity of voltage-gated K+ currents in rat trigeminal ganglion neurons

We investigated the mechanosensitivity of voltage‐gated K+ channel (VGPC) currents by using whole‐cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (IK,A), the other sustained (IK,V). Hypotonic stimulation (200 mOsm) markedly increased both IK,A and IK,V without affecting their activation and inactivation kinetics. Gadolinium, a well‐known blocker of mechanosensitive channels, failed to block the enhancement of IK,A and IK,V induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin‐based cytoskeletal disruptor, further increased IK,A and IK,V, whereas phalloidin, an actin‐based cytoskeletal stabilizer, reduced IK,A and IK,V. Confocal imaging with Texas red‐phalloidin showed that actin‐based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both IK,A and IK,V are mechanosensitive and that actin‐based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons.

Neurochemical Properties of Dental Primary Afferent Neurons

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Aβ fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Aδ fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Aβ fibers. We suggest that these Aβ fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 microM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 microM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca(2+)](i) response; a transient [Ca(2+)](i) peak and sustained [Ca(2+)](i) increase secondary to ATP-stimulated Ca(2+) influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.