The biphasic effect of extracellular glucose concentration on carbachol-induced fluid secretion from mouse submandibular glands.

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca2+ ]i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an ex vivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5 mM and elevating it to higher than 5 mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl- secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca2+ ]i signals.

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

TMEM16E (GDD1) exhibits protein instability and distinct characteristics in chloride channel/pore forming ability.

TMEM16E/GDD1 has been shown to be responsible for the bone-related late-onset disease gnathodiaphyseal dysplasia (GDD), with the dominant allele (TMEM16E(gdd) ) encoding a missense mutation at Cys356. Additionally, several recessive loss-of-function alleles of TMEM16E also cause late-onset limb girdle muscular dystrophy. In this study, we found that TMEM16E was rapidly degraded via the proteasome pathway, which was rescued by inhibition of the PI3K pathway and by the chemical chaperone, sodium butyrate. Moreover, TMEM16E(gdd) exhibited lower stability than TMEM16E, but showed similar propensity to be rescued. TMEM16E did not exhibit cell surface calcium-dependent chloride channel (CaCC) activity, which was originally identified in TMEM16A and TMEM16B, due to their intracellular vesicle distribution. A putative pore-forming domain of TMEM16E, which shared 39.8% similarity in 98 amino acids with TMEM16A, disrupted CaCC activity of TMEM16A via domain swapping. However, the Thr611Cys mutation in the swapped domain, which mimicked conserved cysteine residues between TMEM16A and TMEM16B, reconstituted CaCC activity. In addition, the GDD-causing cysteine mutation made in TMEM16A drastically altered CaCC activity. Based on these findings, TMEM16E possesses distinct function other than CaCC and another protein-stabilizing machinery toward the TMEM16E and TMEM16E(gdd) proteins should be considered for the on-set regulation of their phenotypes in tissues.

Membrane potential modulation of ionomycin-stimulated Ca(2+) entry via Ca (2+)/H (+) exchange and SOC in rat submandibular acinar cells.

Ionomycin (IM) at 5 microM mediates the Ca(2+)/H(+) exchange, while IM at 1 microM activates the store-operated Ca(2+) entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K(+) concentration ([K(+)](o)). IM (5 microM, the Ca(2+)/H(+) exchange) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) to an extremely high value at 151 mM [K(+)](o). However, with increasing [K(+)](o), the rates of Ca(2+) entry decreased in a linear relationship. The reversal potential (E (rev)) for the Ca(2+)/H(+) exchange was +93 mV, suggesting that IM (5 microM) exchanges 1 Ca(2+) for 1 H(+). Thus, depolarization decreases the Ca(2+) influx via the Ca(2+)/H(+) exchange because of its electrogenicity (1 Ca(2+) for 1 H(+)). On the other hand, IM (1 microM, the SOCs) abolished an increase in [Ca(2+)](i) at 151 mM [K(+)](o). With increasing [K(+)](o), the rate of Ca(2+) entry immediately decreased linearly. The E (rev) for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca(2+) over Na(+) (P (Ca)/P (Na) = 8.2). Moreover, an increase in extracellular Ca(2+) concentration (20 mM) enhanced the Ca(2+) entry via the SOCs at 151 mM [K(+)](o), suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca(2+) entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca(2+)](i) via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca(2+)](i) via two pathways depending on its concentration, the exchange of 1 Ca(2+) for 1 H(+) at 5 muM and the SOCs at 1 microM.

Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells.

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.

Suppression of carbachol-induced oscillatory Cl- secretion by forskolin in rat parotid and submandibular acinar cells.

Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca(2+)-activated Cl(-) secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl(-) secretion. Carbachol (Cch), a Ca(2+)-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at -80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca(2+)-activated oscillatory Cl(-) secretion of parotid and submandibular acinar cells at -80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.

Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication.

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.

Irsogladine maleate influences the response of gap junctional intercellular communication and IL-8 of human gingival epithelial cells following periodontopathogenic bacterial challenge.

Gingival epithelial cells first encounter periodontopathogenic bacteria and their metabolic products to produce inflammatory cytokines. Gap junctional intercellular communication (GJIC) is thought to play a critical role in cellular coordination in tissue homeostasis. Gap junctions are structured by connexins (CXs). GJIC response of gingival epithelial cells to the bacteria may be involved in the initiation of periodontal disease. Irsogladine maleate (IM) is known to enhance GJIC through cAMP. In the present study, we examined an effect of IM on GJIC response and on interleukin-8 (IL-8) levels in human gingival epithelial cells (HGEC) exposed to a periodontopathogenic bacterium, Actinobacillus actinomycetemcomitans, and its outer membrane protein (OMP) 29 in order to test the hypothesis that IM has the ability to modulate GJIC and inflammatory responses of gingival epithelial cells to periodontopathogenic bacteria. IM countered the OMP29-induced reduction of GJIC, CX43 levels and cAMP levels in HGEC. The simultaneous addition of OMP29 and dibutyryl cAMP also abrogated the repression of GJIC by OMP29. Furthermore, IM obviated the increase in IL-8 levels in HGEC stimulated by whole live A. actinomycetemcomitans and by OMP29. These findings suggest that IM counters the OMP29-induced GJIC reduction in HGEC through cAMP. IM may eliminate initial perturbation of gingival epithelial cells by regulating responses of GJIC and IL-8 to periodontopathogenic bacterial exposure.

Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells.

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Extracellular proton sensing of the rat gustatory cyclic nucleotide-gated channel.

Elevations of the intracellular levels of cyclic nucleotides appear to cause the cation influx through gustatory cyclic nucleotide-gated (CNGgust) channels expressed in taste cells. Although changes in the oral pH may directly regulate the activity of the CNGgust channel, the mechanism of pH-dependent control of the channel is not understood. In the present study, we combined the whole-cell patch-clamp recording and the site-directed mutagenesis to investigate the effect of extracellular pH on the ion permeation through CNGgust channels expressed in HEK293 cells. Extracellular acidification strongly inhibited ion permeation through open CNGgust channels. Mutation of Glu(289) remarkably attenuated the pH-dependence of the channel, suggesting that Glu(289) in the pore-forming region is a major proton acceptor site. However, the mutant E289A-CNGgust channel possesses the other residual protonation/deprotonation site. The channel activity, tightly regulated by pH(o) and [cNMP](i), suggests the involvement of its pH(o)-dependent ion permeation in taste signal transduction events.

Luminal space enlargement by carbachol in rat parotid intralobular ducts.

Carbachol (CCh) enlarges the luminal space in rat parotid intralobular ducts, but the mechanism of their enlargement remains obscure. We investigated the involvement of intracellular calcium ions in the enlargement of luminal space by monitoring the luminal space under optical sectioning in a confocal laser scanning microscope using sulforhodamine B. Carbachol increased the intracellular concentration of calcium ions ([Ca2+]i) and the inside diameter without any change in the outside diameter. Removal of extracellular calcium ions modulated CCh-induced changes in [Ca2+]i to transient, but did not markedly inhibit the CCh-induced increase in the inside diameter. Additional loading of BAPTA (1,2-bis (o-aminophenoxy-ethane-n,n,n',n'-tetraacetic acid) in the duct cells suppressed CCh-induced changes. Diphenylamine-2-carboxylate (DPC), but not cytochalasin D, calmodulin inhibitor or nitric oxide synthase inhibitor profoundly suppressed CCh-induced changes. These results suggest that CCh induces enlargement of the luminal space through the activation of DPC-sensitive channels by the release of calcium ions from the intracellular pool.

Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate production in rat parotid intralobular ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.

Fluid secretion is observed at the openings of ducts in the exocrine gland. It remains unclear whether the ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the duct lumen by carbachol (CCh) in isolated parotid intralobular duct segments to clarify the ability of the ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the duct lumen were obtained under the optical sectioning at the level of the duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated duct lumen, suggesting that CCh might induce fluid secretion in the duct, leading to the clearance of the dye and small stained clumps from the duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl-, bumetanide or DIDS, suggesting that methazolamide-sensitive production of HCO3-, but not the Cl- uptake mechanism, might contribute to the CCh-induced clearance of the dye from the duct lumen. These results represent the first measurements of fluid movement in isolated duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca2+-activated, DPC-sensitive anion channels with HCO3- secretion in the rat parotid intralobular ducts.