Takako Oshiro

Role of Cyclic ADP-Ribose in ATP-activated Potassium Currents in Alveolar Macrophages

There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells. In the present study, using the nystatin perforated patch recording configuration, we found that ATP (0.3–30 μm) induced a transient outward current in a concentration-dependent manner and that the reversal potential of the ATP-induced outward current was close to the K+ equilibrium potential, indicating that the membrane behaves like a K+ electrode in the presence of ATP. The first application of ATP to alveolar macrophages perfused with Ca2+-free external solution could induce the outward current, but the response to ATP was diminished with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, also diminished the response. When cyclic ADP-ribose (cADPR) was applied to the macrophage cytoplasm, a transient outward current was elicited. Thereafter, the successive outward current was inhibited, suggesting the involvement of cADPR in the response. Intracellular perfusion with inositol 1,4,5-trisphosphate also induced a transient outward current, but the successive current was not inhibited. The ATP-induced outward current was abolished when 8-amino-cADPR (as a blocker of cADPR, 10−6–10−5 m) was introduced into the cytoplasm. Homogenates of alveolar macrophages showed both ADP-ribosyl cyclase and cADPR hydrolase activities, and CD38 (ADP-ribosyl cyclase/cADPR hydrolase) expression was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These results indicate that ATP activates K+currents by releasing Ca2+ from cADPR-sensitive internal Ca2+ stores.

Differential effect of epidermal growth factor on serous and mucous cells in porcine airway submucosal gland.

Using a patch-clamp technique, we found that the fresh porcine submucosal gland acinar cells contained two functionally distinct cell populations, i.e. physiologically relevant concentration of acetylcholine (ACh, 30 nM) induced two distinct patterns of electric response in tracheal gland acinar cells. One was characterized by an outstanding oscillatory Cl(-)-current activity, and the other was with poor Cl(-)-current response but with a comparable K(+)-current. We examined the effect of epidermal growth factor (EGF) on the ACh-induced electric responses in these cells. EGF affected only the latter (K(+)-prominent) cell type to potentiate significantly the ACh-induced K(+)-current. An immunohistochemistry revealed that the receptor for EGF was identified preferentially on the mucous, but not serous, cells. Genistein, one of the tyrosine-kinase inhibitors, abolished the augmentation effect of EGF on the ACh-induced current. Thus, we identified the serous cell with a Cl(-)-rich current in response to ACh and the mucous cell with a K(+)-dominant response. Moreover, EGF affected the mucous cells alone to potentiate the ACh-induced electric response. EGF may contribute to the pathophysiological alterations in chronic inflammatory airways both in morphological (mucous cell hypertrophy/hyperplasia) and functional (thick viscous hypersecretion) ways.

Cholinomimetic action of macrolide antibiotics on airway gland electrolyte secretion

We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl- currents were reduced to 54 and 83% and the outward K+ currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+ channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl- secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl-currents were reduced to 54 and 83% and the outward K+ currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+ channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl- secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.

Red wine polyphenols and swallowing reflex in dysphagia

Aspiration is the most life-threatening medical problem followed by dysphagia, which is experienced by approximately 50% of patients in nursing homes. Dysphagia is caused not only by a motor impairment, but also by an impairment of the sensory (neural) component of swallowing; that is, an impairment of the swallowing reflex. Triggering of the swallowing reflex can be accelerated if swallowed material is warmed up, even in dysphagic patients. Furthermore, it has been reported that capsaicin and capsiate, transient receptor potential vanilloid 1 (TRPV1) agonists, accelerated the swallowing reflex in patients with aspiration pneumonia. Because a hot sensation is sensitized through TRPV1, the results show that TRPV1 stimulation, together with pharyngeal food stimuli, accelerate the swallowing reflex in the elderly. Twenty-five hundred years before the beneficial effect of red wine in reducing the incidence of cardiovascular disease became widely known as the “French Paradox”, Hippocrates, the Greek physician, had already used red wine not only as a medicine for a variety of ailments, such as diarrhea, difficult childbirth and lethargy, but also as a disinfectant, an aid to digestion, a diuretic and as a carrier of other drugs. Here, we report our successful use of red wine as a treatment for dysphagia and also an examination of the cellular mechanism from which this beneficial effect of red wine polyphenolic compounds (RWPC) is derived. We assessed the swallowing reflex in all residents (mean age 84 years [SD 7], n = 42) in a nursing home located in a suburb of Sendai, Japan. Written informed consent was obtained from every subject. The swallowing reflex was provoked by a 1 mL of bolus solution injected into the pharynx through a nasal catheter. The swallowing reflex was quantified from the injection to the onset of swallowing. In the nursing home, 14 elderly patients with episodes of dysphagia (mean age 86 years [SD 6]) suffered from swallowing reflex impairment (latency >5 s with distilled water). Computed tomography showed various degrees of cerebral atrophy and lacunar infarction in these patients. We assessed the swallowing reflex with various stimuli in these 14 elderly patients. Distilled water, red and white wines without alcohol, and red grape juice were applied at intervals of 2 min in a double-blind and randomized manner. A red wine made from Cabernet Sauvignon grapes and a white wine made from Koshu grapes were evaporated under reduced pressure (40°C) to half the original volume in order to remove the ethanol. Red grape juice made from Concord red grapes was not concentrated. The latency of response to red wine was significantly shorter than those of vehicle, white wine and red grape juice (P < 0.001 versus the others by ANOVA with a post-hoc application of Fisher’s LSD test; Fig. 1a). Then, we randomly selected seven subjects from the 14 subjects by the envelope method. To confirm the effect of RWPC on improving the latency, we applied purified RWPC to these seven subjects in a double-blind manner. RWPC extracted from Cabernet Sauvignon grapes (>90% purity) was quantified by the Folin–Ciocalteu method (83.3% Gallic acid equivalent). RWPC was dissolved in distilled water at various concentrations. The latency decreased as a function of RWPC (Fig. 1b), suggesting that RWPC facilitated the swallowing reflex in these elderly patients. The rapid effect of RWPC suggests the existence of a neural modification of the swallowing reflex by RWPC. Because the swallowing reflex is mediated by a capsaicinsensitive neural pathway, we postulated that the molecular basis for the effect of RWPC is at least partly attributed to activation of TRPV1, the capsaicin receptor. Therefore, we investigated the direct interaction of RWPC and the capsaicin-induced response using mouse dorsal root ganglion (DRG) neurons with TRPV1 as a model system. DRG neurons of adult ddY mice (8 weeks) were enzymatically dissociated in Leibovitz’s L-15 Medium (LLM) supplemented by 3 mg/mL collagenase medium at 37°C for 20–30 min. Using a conventional Geriatr Gerontol Int 2010; 10: 329–330

Delayed expression of large conductance K+ channels reshaping agonist‐induced currents in mouse pancreatic acinar cells

Epithelial secretory cells display cell‐specific mechanisms of fluid secretion and express large conductance voltage‐ and Ca2+‐activated K+ (Maxi‐K) channels that generate the membrane negativity for effective Cl− exit to the lumen. Rat and mouse pancreatic acinar cells had been thought to be peculiar in this sense because of the previously reported lack of Maxi‐K channels. However, this view is not entirely correct as evidenced in the present paper. Searching for their presence in pancreatic acinar cells in mice from 5 to 84 weeks of age with patch‐clamp current measurements, we demonstrated that the expression of Maxi‐K channels is regulated in an age‐associated manner after birth. The expression started at approximately 12 postnatal weeks and increased steadily up to 84 weeks. In support of this, RT‐PCR could not detect mSlo mRNA, the Maxi‐K gene, at either 7 or 8 weeks but could at 58 and 64 postnatal weeks. These results suggest that a key steering element for fluid secretion, the Maxi‐K channel, is progressively re‐organized in rodent pancreas. A pancreatic secretagogue, acetylcholine, evoked Maxi‐K channel current overlapping to various degrees on the previously known current response. This suggests that the rise in internal Ca2+ activates Maxi‐K channels which reshape the mode of secretagogue‐evoked current response and contribute to Cl− driving in fluid secretion in an age‐associated fashion.

Involvement of GTP-binding protein in pancreatic cAMP-mediated exocytosis

Abstract.We studied cAMP-mediated exocytosis in rat pancreatic acinar cells. We monitored changes in the membrane capacitance (ΔC), which reflects the granule fusion/retrieval process, with whole-cell patch-clamp capacitance measurement. The rise in cellular cAMP, caused indirectly by receptor activation by vasoactive intestinal polypeptide or directly by dibutyryl cyclic AMP, was able to induce an increase in ΔC independently of cellular Ca2+. Using the latter stimulation, we estimated the magnitude of the response to internal GTPγS [guanosine 5′-(γ-thio)trisphosphate] and/or GDPβS [guanosine 5′-(β-thio)diphosphate]. The internal GTPγS and GDPβS amplified and depressed the response, respectively. Thus, the cellular cAMP alone can trigger granule insertion independently of cellular Ca2+ and it can be controlled by cellular GTP-binding proteins, presumably those belonging to the Rab family.