Hideyo Yoshida

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video‐optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2‐methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 μm, an inhibitor of purinergic receptor). Ionomycin (5 μm) or thapsigargin (2 μm) increased CBF similarly. Ca2+‐free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 μm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+‐free solution decreased CBF, and ionomycin (5 μm) or thapsigargin (2 μm) increased it. Moreover, acetylcholine (100 μm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 μm), a selective β2‐adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+‐mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.

Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons.

PURPOSE To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death. METHODS Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels ([Ca(2+)](i)) were examined. RESULTS Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in [Ca(2+)](i), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP. CONCLUSIONS The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.

[Ca2+]i Oscillations Induced by High [K+]o in Acetylcholine‐Stimulated Rat Submandibular Acinar Cells: Regulation by Depolarization, cAMP and Pertussis Toxin

Maintaining the extracellular K+ concentration ([K+]o) between 15 and 60 mM induced oscillations in the intracellular Ca2+ concentration ([Ca2+]i) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 μM). These [Ca2+]i oscillations were also induced by 1 μM thapsigargin and were inhibited by 50 μM La3+, 1 μM Gd3+, or the removal of extracellular Ca2+, indicating that the [Ca2+]i oscillations were generated by store‐operated Ca2+ entry (SOC). The frequency of the ACh‐evoked [Ca2+]i oscillations increased from 0.8 to 2.3 mHz as [K+]o was increased from 15 to 50 mM. TEA (an inhibitor of K+ channels) also induced [Ca2+]i oscillations at [K+]o of 4.5 or 7.5 mM in ACh‐stimulated cells. These data suggest that depolarization causes [Ca2+]i to oscillate in ACh‐stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca2+]i to be sustained at a high level in ACh‐stimulated cells at 25 mM or 60 mM [K+]o. This suggests that the [Ca2+]i oscillations are generated by a periodic inactivation of the SOC channels via PTX‐sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca2+]i oscillations remained constant (approximately 1.2 mHz) when [K+]o was maintained in the range 25‐60 mM. Based on these observations in ACh‐stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX‐sensitive G proteins, which inactivate the SOC channels periodically ([Ca2+]i oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX‐sensitive G proteins.

Hypo‐osmotic potentiation of acetylcholine‐stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video‐enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca2+ concentration ([Ca2+]i) in a dose‐dependent manner. An adequate hypo‐osmotic stress (−40 mosm) potentiated ACh‐stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo‐osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo‐osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo‐osmotic potentiation of ACh‐stimulated CBF increase and that ATP increased [Ca2+]i and CBF, as well as potentiating ACh‐stimulated rises in [Ca2+]i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X4 purinergic receptor. A hypo‐osmotic stress (−40 mosM) transiently increased [Ca2+]i and potentiated the ACh‐stimulated [Ca2+]i increase. The hypo‐osmotic potentiation of ACh‐stimulated CBF increase was not detected under Ca2+‐free conditions. These observations suggest that a hypo‐osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca2+]i and CBF in ACh‐stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X4.

Cell shrinkage evoked by Ca2+‐free solution in rat alveolar type II cells: Ca2+ regulation of Na+–H+ exchange

The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT‐II cells) were examined. Perfusion with a Ca2+‐free solution induced shrinkage of the AT‐II cell volume in the absence or presence of amiloride (1 μm, an inhibitor of Na+ channels); however, it did not in the presence of 5‐(N‐methyl‐N‐isobutyl)‐amiloride (MIA, an inhibitor of Na+–H+ exchange). MIA decreased the volume of AT‐II cells. Inhibitors of Cl−–HCO3− exchange, 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonic acid (SITS) also decreased the volume of AT‐II cells. This indicates that the cell shrinkage induced by a Ca2+‐free solution is caused by a decrease in NaCl influx via Na+–H+ exchange and Cl−–HCO3− exchange. Addition of ionomycin (1 μm), in contrast, induced cell swelling when AT‐II cells were pretreated with quinine and amiloride. This swelling of the AT‐II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+‐free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA‐treated AT‐II cells. Ionomycin increased [Ca2+]i in fura‐2‐loaded AT‐II cells. In conclusion, the Na+–H+ exchange activities of AT‐II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.

Osmotic flow transients during acetylcholine stimulation in the perfused rat submandibular gland.

Osmotic stress was applied to the perfused rat submandibular gland during steady‐state fluid secretion. Alterations of perfusate osmolarity, by addition or withdrawal of sucrose or NaCl, caused transient changes in secretory rate during continuous stimulation with 1 microM acetylcholine (ACh). Hyposmotic perfusates transiently increased, and hyperosmotic perfusates transiently reduced, the secretory rate. The transients were attributed to changes in osmotic flow resulting from changes in the instantaneous transepithelial osmotic gradient. The time course of the change in interstitial osmolarity was determined by using a Cl‐ electrode to record the changes in interstitial Cl‐ concentration following a step change in perfusate Cl‐ concentration. From the calculated changes in interstitial osmolarity and the resulting changes in secretory rate, the osmotic water permeability of the secretory pathway was estimated to be greater than 15.0 +/− 1.2 microliter (mosmol 1‐1)‐1 min‐1 (g wet weight)‐1 (9.8 x 10(‐6) +/− 0.8 x 10(‐6) l atm‐1s‐1g‐1). The transepithelial gradient required to sustain steady state, ACh‐evoked secretion would therefore be less than 16 mosmol l‐1 NaCl, which is consistent with previous micropuncture data indicating that the luminal fluid is approximately isosmotic.

Transepithelial fluid shift generated by osmolarity gradients in unstimulated perfused rat submandibular glands

The effects of osmotic gradients on transepithelial water movements were examined in unstimulated perfused submandibular glands of the rat. Osmotic gradients were applied transepithelially by adding sucrose to or removing it from the perfusate. An infusion of hypotonic perfusate shifted fluid from the interstitium to the lumen (luminal fluid shift) transiently, whereas an infusion of hypertonic perfusate shifted fluid from the lumen to the interstitium (interstitial fluid shift) transiently. The amount of fluid shifted from lumen to interstitium increased as the luminal fluid osmolarity was raised or as the perfusate osmolarity was reduced. Thus, fluid movements across the salivary epithelium were shown to be simply dependent on the osmolarity difference between lumen and interstitium. To estimate the effective pore radius of the epithelium, non‐electrolyte solutions (urea, dimethylurea, diethylurea, mannitol, sucrose and maltotriose) were also used as luminal solutions. The results from non‐electrolyte experiments showed that the effective pore radius of the passage for non‐electrolytes was slightly larger than 0.38 nm. Solutes smaller than mannitol were less effective in opposing the interstitial fluid shift, and the value of effective pore radius in this report was similar to that of the secretory water pathway that has been measured in solvent drag studies (0.4 0.45 nm). These findings suggest that the passage for non‐electrolytes may be water transport pathway in salivary epithelium.

Network synthesis of the epithelial transport system

Abstract The epithelial transport system consists of structural subsystems of three transport barriers (membranes) and three solution compartments. Macroscopically, coupled dissipations in the system occur in barrier subsystems, and coupled free-energy increasing or decreasing processes in the system occur in compartment subsystems. Therefore, the barrier subsystem is assumed to be the dissipating subsystem, and the compartment subsystem is assumed to be the free-energy changing subsystem. These structural subsystems consist also of a set of elemental thermodynamic processes, that is, dissipations, processes of free-energy change and power conversions. Each elemental process can be represented by thermodynamic elements prepared in bond graphs. p]In the experiments described in this paper, thermodynamic elements expressing free-energy changes and couplings in compartment subsystems were gathered and modeled as a capacitive module. The capacitive module behaved as a solution compartment and a simultaneous equation could be derived in which a set of flows determined a set of absolute-potential changes in the solution compartment. Elements were gathered and were modeled as a resistive module. The resistive module behaved as a membrane and a simultaneous transport equation could be derived in which a set of forces determined a set of flows through the membrane. A model of the epithelial transport system was achieved by the block diagram with the above modules. As variables in the module, the force vector and the flow vector expressing sets of power variables were assigned, but not a force nor a flow. This model showed the topology of the system and behaved as the real system. This model and the derived equations were used to simulate the frog skin behavior as an example of an epithelial transport system.

{\text{HCO}}^{{\text{ - }}}_{{\text{3}}}-dependent pHi recovery and overacidification induced by {\text{NH}}^{ + }_{4} pulse in rat lung alveolar type II cells: {\text{HCO}}^{ - }_{3} -dependent NH3 excretion from lungs?

Intracellular pH (pHi) after the