A role of sodium bicarbonate cotransporter(NBC) in HCO3- formation in human salivary gland acinar cells

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in HCO3- formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) -loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (△pHi/min) after cell acidification using an ammonium prepulse technique. In control experiments, △pHi/min was 1.40 ± 0.06. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced △pH/min to 1.14 ± 0.14 and 0.74 ± 0.15, respectively. Simultaneous application of DIDS and acetazolamide further reduced △ pHi/min to 0.47 ± 0.10. Therefore, DIDS and acetazolamide reduced △pHi/min by 19 % and 47 %, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in △pHi/min of 67 %. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to HCO3- formation in human parotid gland acinar cells.This work was supported by the Korea Science & Engineering Foundation (KOSEF) grant funded by Korea government (R13-2008-008-01001-0) through the Oromaxillofacial Dysfunction Research Center for the Elderly(ODRCE) at Seoul National University

Characterization of Intermediate Conductance K+ Channels in Submandibular Gland Acinar Cells

There are some evidences that K+ efflux evoked by muscarinic stimulation is not mainly mediated by large conductance K+ (BK) channels in salivary gland. In this experiment, we therefore characterised non BK channels in rat submandibular gland acinar cells and examined the possibility of agonist effect on this channel using a patch clamp technique. Two types of K+ channels were observed in these cells. BK channels were observed in 3 cells from total 6 cells and its average conductance was 152±7 pS (n=3). The conductance of the another types of K+ channel was estimated as 71±7 pS (n=6). On the basis of the conductance of this channel, we defined this channel as intermediate conductance K+ (IK) channels, which were observed from all 6 cells we studied. When we increased Ca2+ concentration of the bath solution in inside-out mode, the IK channel activity was greatly increased, suggesting this channel is Ca2+ sensitive. We next examined the effect of carbachol (CCh) and isoproterenol on the activity of the IK channels. 10-5 M isoproterenol significantly increased the open probability (Po) from 0.08±0.02 to 0.21±0.03 (n=4, P＜0.05). Application of 10-5 M CCh also increased Po from 0.048±0.03 to 0.55±0.33 (n=5, P＜0.05) at the maximum channel activity. The degree of BK channel activation induced by the same concentration of CCh was lower than that of IK channels; Po value was 0.011±0.003 and 0.027±0.005 in control and during CCh stimulation (n=3), respectively. The result suggests that IK channels exist in salivary acinar cells and its channel activity is regulated by muscaricinic and β- adrenergic agonist. We conclude that IK channels also play a putative role in secretion as well as the BK channels in rat submandibular gland acinar cells.This work was supported by a grant of the Korean Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (Grant No 00-PJ1-PG1-CH10-0002)

Double-stranded RNA induces inflammatory gene expression in schwann cells: Implication in the Wallerian degeneration

Schwann cells play an important role in peripheral nerve regeneration. Upon neuronal injury, activated Schwann cells clean up the myelin debris by phagocytosis, and promote neuronal survival and axon outgrowth by secreting various neurotrophic factors. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that certain cytoplasmic molecules, which are secreted by cells undergoing necrotic cell death, induce immune cell activation via the toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. Accordingly, this study was undertaken to examine the expression and the function of the TLRs on primary Schwann cells and iSC, a rat Schwann cell line. The transcripts of TLR2, 3, 4, and 9 were detected on the primary Schwann cells as well as on iSC. The stimulation of iSC with poly (I：C), a synthetic ligand for the TLR3, induced the expression of TNF-α and RANTES. In addition, poly (I：C) stimulation induced the iNOS expression and nitric oxide secretion in iSC. These results suggest that the TLRs may be involved in the inflammatory activation of Schwann cells, which is observed during Wallerian degeneration after a peripheral nerve injury.This work was supported by the Korea Research Foundation Grant (KRF-2003-003-E00178)

Expression of volume-activated anion channels in exocrine acinar cells

Volume-sensitive anion channels (Iclswell)are expressed in most mammalian cells (1). The molecular identity of Iclswell is not known, however, several candidate proteins have been proposed including: p-glycoprotein, pIcln, CIC-2 and CIC-3(2). The properties of CIC-3 make it one of the most likely candidate proteins, e.g. it has a structure which is very similar to that of know Cl- channels (CIC-0 and CIC-1), and it produces an outward-rectifying Cl- conductance when expressed in Xenopus oocytes or mammalian cell lines (3)

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis

Effects of various neurotransmitters on the intracellular calcium concentration in trigeminal ganglion neurons

Functional neurotransmitter receptors which elevate intracellular free calcium concentration ([Ca2i]j have been identified in freshly isolated trigeminal ganglion (TG) neurons in neonatal rat pups. Measurement of [Ca2´], was performed using fura-2 acetoxymethyl ester (Flea-2) based ratio microspectrofluorimetry. In our experiments only small sized neurons (<30 m) according to somatic diameter were used. In single intact neurons, the expression percentage and magnitudes of [Ca´+], increase responding to each agonist were determined. Application of bradykinin (BK), substance P (SP), calcitonin gene related peptide (CGRP), ATP and capsaicin elevated [Ca"], in the expression percentages of 24%, 37%, 31%, 77% and 50%, respectively, and elevated [Ca 2´], from 1.630.04 (MeanS.E) to 1.920.05, from 1.360.08 to 1.880.12, from 1.33 0.03 to 1.610.09, from 1.350.04 to 2.20.15 and from 1.260.06 to 2.90.26, respectively. The purinergic receptor which respond to ATP was most frequently expressed (77%). A [Ca`], increase in response to all neurotransmitters was transient in nature, except in response to capsaicin. Increase of [Ca2i], evoked by capsaicin was not transient: [Ca2´], was maintained at a higher concentration than the prestimulus level. The magnitude of [Ca 2´], increase evoked by capsaicin was bigger than for any other neurotansmitters. Our results show that a subpopulation of trigeminal ganglion neurons with small diameter express functional receptors responsible for elevating [Ca2i], coexisting with other neurotransmitter receptors in the early neonatal stage. Our data may have implications for studying orofaciai pain transmission which is associated with intracellular calcium increase

Identification and regulation of K+ and Cl- channels in human parotid acinar cells

The properties of K+ channels in these cells were studied using patch-clamp methods. Two channels, with conductances of 165±13 pS (n=6) and 30±1 pS (n=3), were identified in single-channel experiments. In cell-attached patches the reversal potentials were −67±8 and −74±2 mV for the large and small conductance channel, respectively, suggesting that both channels are K+-selective. The large conductance channel was also shown to be K+-selective in inside-out patches. The open probability (Po) of this channel was increased at depolarizing potentials and by increasing intracellular Ca2+ concentration ([Ca2+]i). These properties suggest that the large conductance channel is a maxi Ca2+-activated K+ channel (BKCa). The small conductance channel was not observed in inside-out patches. Carbachol (CCh; 10−5 M) activated the BKCa channel, but not the small conductance channel, in cell-attached patches. CCh also caused a dose-dependent increase in [Ca2+]i measured by fura-2 in microspectrofluorimetric studies, with a half-maximal response at approximately 3×10−6 M. Neither isoproterenol (10−5 M) nor substance P (10−6 M) affected K+-channel activity or [Ca2+]i. In whole-cell experiments, CCh caused an increase in outward current. Charybdotoxin (10−7 M), a BKCa blocker, inhibited a large component of the CCh-induced current. A large component of the charybdotoxin-insensitive current may be carried by Ca2+-activated Cl− channels, which were also observed in human parotid acinar cells. The results indicate that BKCa channels make a significant contribution to the whole-cell conductance in human parotid acinar cells