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The Th2 cytokines, interleukin (IL)-4 and IL-13, bind to IL-4R, that IL-4 inhibited mucus secretion and attenuated the gene and cause goblet cell metaplasia/hyperplasia with increased expression of MUC5AC and MUC5B in primary human mucin expression in vivo. However, there is not enough evi- bronchial epithelial cells (8), and that IL-4 did not induce dence that these cytokines directly induce mucin production in MUC5AC gene expression in mucoepidermoid carcinoma vitro. In this study, primary epithelial cells from guinea pig cell line, NCI-H292 (9). Concerning IL-13, Longphre and cotrachea were cultured at an air–liquid interface, and immedi- workers (10) reported that IL-13 did not increase MUC5AC ately after achieving confluence at Day 7 they were treated gene expression in NCI-H292 cells, but it is uncertain with human recombinant IL-4 or IL-13 for 14 d. IL-13–treated whether IL-13 induces mucin production and MUC5AC cells consisted of a large number of fully mature goblet cells expression in primary culture of lower airway epithelium. with a smaller number of ciliated cells. Secretory granules of the goblet cells were positive for both periodic acid-Schiff and We studied the effects of IL-4 and IL-13 on airway epitoluidine blue, and showed exocytosis. By contrast, IL-4 failed thelial differentiation using air–liquid interface culture, a to induce goblet cell differentiation. The electric resistances of procedure that induces high levels of differentiation (11–13), IL-13–treated cells were lower than those of IL-4–treated cells with airway epithelial cells differentiating into both ciliated and nontreated cells, suggesting leaky epithelia. MUC5AC pro- and goblet cells. In this study, we focused on the numbers of tein level in cell lysates measured by ELISA was several-fold goblet cells and ciliated cells, because goblet cell hyperplasia higher in IL-13–treated cells than in nontreated cells, whereas along with correspondingly fewer ciliated cells may cause the level in IL-4–treated cells was not changed. These data the impairment of mucociliary clearance that is found in suggest that human recombinant IL-13, but not IL-4, can induce the airways of patients who die of asthma (14). We also

Arginine vasopressin (AVP) has recently been shown to exist in and to be released from airway epithelial cells, but the physiologic role of this hormone in airway epithelial function is unknown. To determine whether AVP affects ciliary motility, and if so, to elucidate the mechanism of action and the subtype of AVP receptors involved, we measured ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) of cultured rabbit tracheal epithelium with a photoelectric method and the fura-2 fluorescence method, respectively. Addition of AVP caused a rapid increase in CBF, followed by a decline and a subsequent sustained response. The ciliary stimulatory action was dose dependent, the maximal peak increase from the baseline CBF being 20.6 +/- 4.7% (mean +/- SE, P0.001), and this effect was reduced to 5.9 +/- 2. 0% by the V1 receptor antagonist OPC-21268 (P0.01), but not by the V2 receptor antagonist OPC-31260. The AVP-induced increase in CBF was not altered by the protein kinase A (PKA) inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate triethylamine (Rp-cAMPS) or by Ca2+-free solution containing ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), but was abolished by pretreatment with thapsigargin. Exposure of cells to AVP elicited a transient increase in [Ca2+]i, an effect that was likewise abolished by thapsigargin. The rank-order potency of AVP analogues to increase [Ca2+]i was AVP = [deamino1, D-3-(pyridyl) Ala2-Arg8] vasopressin (DP-VP), a specific V1b receptor agonist[Phe2, Ile3, Orn8] vasopressin (PO-VT), a V1a agonist1-desamino-8-D-arginine vasopressin (dDAVP), a V2 agonist. Moreover, OPC-21268 greatly attenuated the action of AVP, whereas OPC-31260 was without effect. These results suggest that AVP stimulates ciliary motility of rabbit tracheal epithelium through mobilization of Ca2+ from thapsigargin-sensitive stores, and that this effect may be mediated by V1b receptors.

The effect of platelet activating factor (PAF) on the intracellular cytosolic levels of free calcium ([Ca2+]i) was studied in cultured epithelium from cow trachea. In fura-2-loaded cells, PAF (10(-9) to 10(-5) M), but not lyso-PAF, increased [Ca2+]i in a concentration-dependent manner, from 106 +/- 15 to 270 +/- 40 nM (P0.05). This [Ca2+]i response consisted of a transient increase that peaked within 15 s after addition and a subsequent sustained elevation that reached a plateau after 1 min. The potency for the sustained response was greater by approximately 1 log U than that for the transient response. Preincubation of the cells with the PAF receptor antagonist CV6209 (10(-6) M) inhibited the increase in [Ca2+]i. Ca(2+)-free medium (2 mM EGTA) totally abolished the sustained response to PAF, but it only partially inhibited the transient response. Verapamil (10(-5) M) also largely inhibited the sustained response. Moreover, PAF transiently increased inositol triphosphate (IP3) levels, peaking 10 s after addition. These data suggest that (1) a PAF-induced increase in [Ca2+]i is mediated via PAF receptors, (2) PAF causes both transient [Ca2+]i release from intracellular stores through IP3 production and sustained [Ca2+]i influx from extracellular solution, and (3) Ca2+ influx may be a major pathway of PAF-induced increase in [Ca2+]i in cow tracheal epithelium.

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.

Erythromycin (EM) therapy is known to decrease airway secretion in chronic inflammatory airway diseases such as diffuse panbronchiolitis. Airway secretion is regulated by intracellular Ca2+ concentration ([Ca2+]i). To elucidate the intracellular site of action of EM in airway epithelium, we examined the effect of EM on Ca2+ dynamics in cultured bovine tracheal epithelial cells using fura-2. EM per se did not cause any change in [Ca2+]i. Adenosine triphosphate (ATP; 10(-4) M) induced a biphasic [Ca2+]i increase, consisting of a transient response followed by a sustained response. Pretreatment of cells with EM had little effect on the ATP-induced transient Ca2+ response but substantially reduced the sustained response in a dose-dependent manner. Clarithromycin, another 14-membered ring macrolide, likewise showed the inhibitory effect, but ampicillin and cephasolin did not. Uridine triphosphate (UTP; 10(-4) M) induced a biphasic [Ca2+]i increase similar to ATP, and the UTP-induced sustained Ca2+ response was also inhibited by EM. In Ca2+-deficient medium (1 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N, N'-tetraacetic acid [EGTA]) or in the presence of La3+, the sustained Ca2+ response disappeared, suggesting that EM may inhibit Ca2+ influx induced by P2u purinoceptor stimulation. In single-cell Ca2+ image analysis, low concentration of ATP (10(-6) M) induced Ca2+ oscillations, which were also inhibited by EM. The disappearance of [Ca2+]i oscillations after addition of EM was similar to that after addition of EGTA. These results suggest that EM may decrease Ca2+-dependent airway secretion by inhibiting agonist-stimulated Ca2+ influx.

Airway epithelial cells cultured at the air-liquid interface possess highly differentiated functions and structures compared with the cells cultured under immersion. We examined the oxidative metabolism and glycolysis in cow tracheal epithelial cells on Days 3, 6, 10, and 13, cultured under three different conditions: (1) immersion culture on porous filters with apical and basolateral feeding (IM), (2) air-exposed culture on porous filters with basolateral feeding, i.e., air-liquid interface culture (AI), and (3) conventional immersion culture in plastic dishes with apical feeding (DI). Lactate production was less in AI than in IM and DI on Day 3 through Day 13, whereas cellular adenosine triphosphate content and basal O2 consumption were greater. Ouabain-sensitive and ouabain-insensitive O2 consumption, and the uncoupled O2 consumption were also greater in AI. Cytosolic lactate dehydrogenase activities on Day 10 were lower in AI, whereas alpha-ketoglutarate dehydrogenase activities were higher. The increased oxidative metabolism in AI was more pronounced at the late phase of culture (Days 10 and 13). In contrast, glycolysis remained elevated during the experiment in IM and DI. These data suggest that (I) AI begins to promote oxidative metabolism from growth phase by the provision of adequate oxygenation, and then further shifts to oxidative metabolism with differentiation; and (2) apical feeding may be responsible for the disturbance of the development of the oxidative metabolism.