Chikara Hirono

Induction of muscarinic cholinergic responsiveness inXenopus oocytes by mRNA isolated from rat brain

Microinjection of mRNA from adult rat brains into Xenopus oocytes induced an electrophysiological response to acetylcholine in the cells. The cells responded to acetylcholine through muscarinic receptors which appeared on the cell surface after microinjection of the mRNA and showed the depolarization, mainly due to an increase in membrane permeability to Cl ions. The acetylcholine reaction did not require extracellular Ca ions and was suppressed by pertussis toxin, suggesting the involvement of the inhibitory guanine nucleotide-binding regulatory protein.

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Abstract. Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K+ and Cl− currents (IK and ICl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca2+ concentration ([Ca2+]i), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn2+ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca2+]i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of IK and ICl mainly by the increased cyclic AMP level and partially by the potentiated Ca2+ influx and [Ca2+]i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca2+]i increase.

Characterization of mRNA responsible for induction of functional sodium channels in Xenopus oocytes

When Xenopus laevis oocytes were microinjected with poly(A)+ mRNA isolated from adult rat brains or electric organs of Electrophorus electricus, the oocytes developed functional sodium channels. Upon application of veratrine, the microinjected oocytes exhibited transient depolarization, resulting in spontaneous repetitive spikes in some occasions, and action potentials. These responses were mediated mainly by external Na ions, prolonged by scorpion toxin, completely blocked by tetrodotoxin, and suppressed by local anesthetics. Thus the mRNA-induced sodium channels exhibited essentially all the functional properties expected for native sodium channels in nerve and muscle membranes. Rat brain mRNA was fractionated into 4 fractions by sucrose gradient centrifugation. Each fraction and various combinations of them were examined for the efficiency in inducing functional sodium channels in Xenopus oocytes. A fraction corresponding to mRNA of approximately 30S to 46S was found to contain all mRNA necessary for the expression of the channels, indicating that mRNA of smaller sizes expected to code for smaller polypeptides may not be required.

Characterization of glutamate receptors induced in Xenopus oocytes after injection of rat brain mRNA.

Xenopus oocytes in which poly(A)+ mRNA isolated from rat brains were previously injected, exhibited at least 3 categories of current responses to excitatory amino acids. They were oscillatory responses to glutamate (Glu) or quisqualate (QA), smooth large responses to kainate (KA), and smooth small responses to Glu and QA. Oscillatory responses were mediated by a metabotropic type of Glu receptor which is coupled to a G-protein but not directly to an ionic channel. Amplitudes of smooth Glu responses and smooth QA responses were similar in size, and were not additive to each other, suggesting a common receptor mediating both responses. L-Glutamylglycine inhibited KA responses in a competitive manner without affecting smooth Glu/QA responses, indicating that KA and smooth Glu/QA responses were mediated by separate receptors. From these results, it was concluded that the injection of rat brain mRNA induced at least 3 different glutamatergic receptors: receptors mediating (a) KA responses and (b) smooth Glu/QA responses, and (c) the metabotropic Glu receptor. The former two may most likely correspond to Glu receptor subtypes preferring KA and QA, respectively, as seen in the brain.

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 (TMEM16Egdd) 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, TMEM16Egdd 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 TMEM16Egdd proteins should be considered for the on‐set regulation of their phenotypes in tissues. J. Cell. Physiol. 229: 181–190, 2014.

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Abstract. Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3− secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3−. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3− significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and production of cytosolic HCO3− induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3− exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3− exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3− produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

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.

Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory Cl− Movements in Salivary Acinar Cells

Elevations of cytoplasmic free calcium concentrations ([Ca2+]i) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl− through Ca2+-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−-HCO3 − 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 HCO3 − had significant effects, suggesting that the cotransporter rather than parallel operations of Cl−-HCO3 − and Na+-H+ exchangers is the primary Cl− uptake pathway. Pharmacological manipulation of the activities of the Ca2+-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 Ca2+-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.

Irsogladine maleate counters the interleukin‐1β‐induced suppression in gap‐junctional intercellular communication but does not affect the interleukin‐1β‐induced zonula occludens protein‐1 levels in human gingival epithelial cells

BACKGROUND AND OBJECTIVE Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1 beta is involved in periodontal disease. Little is known, however, about the effect of interleukin-1 beta on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1 beta. In this study, we examined how interleukin-1 beta affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1 beta-induced changes in the intercellular junctional complexes in human gingival epithelial cells. MATERIAL AND METHODS Human gingival epithelial cells were exposed to interleukin-1 beta, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. RESULTS Interleukin-1 beta decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1 beta-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. CONCLUSION The effect of interleukin-1 beta on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis.

Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA☆

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serum-treated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.