Noritaka Sakai

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.

Effects of platelet-activating factor on bioelectric properties of cultured tracheal and bronchial epithelia

To elucidate the effect of platelet-activating factor (PAF) on ion transport function of airway epithelial cells, we studied bioelectric properties of cultured tracheal and bronchial epithelia from dogs under short-circuit conditions in vitro. Addition of PAF (10(-5) mol/L) to mucosal solution of Ussing chamber increased short-circuit current of tracheal epithelium from 3.3 +/- 0.7 to 8.5 +/- 1.2 microA/cm2 (p less than 0.001). This effect was dose dependent, and there was a corresponding increase in transepithelial potential difference. In contrast, PAF was without effect when it was added to the submucosal side. Electrical properties of bronchial epithelium remained unchanged by PAF. The PAF-induced increase in short-circuit current was not affected by amiloride but abolished by diphenylamine-2-carboxylate, bumetanide, or Cl-free medium. The effects of PAF were not altered by AA-861 or U-60257, but attenuated by indomethacin and piroxicam, and dose-dependently blocked by CV 6209 and WEB 2086. Mucosal, but not submucosal, addition of PAF increased the rate of prostaglandin release from tracheal epithelium. These results suggest that PAF selectively stimulates Cl secretion across tracheal epithelium, probably through activation of its specific receptors and the subsequent production of prostaglandins.

Stimulation of ciliary motility mediated by atypical β-adrenoceptor in canine bronchial epithelium

The effects of catecholamines on ciliary motility of canine bronchial epithelium was studied by a photoelectric method in vitro. Addition of beta-adrenoceptor agonists increased ciliary beat frequency, a rank order of potency being salbutamol > or = BRL 37344, a selective beta 3-adrenoceptor agonist > norepinephrine. The response to BRL 37344 was relatively resistant to the blockade of beta 1- and beta 2-adrenoceptors but was competitively antagonized by the beta 3-adrenoceptor antagonist cyanopindolol, with the pA2 value being lower than that when salbutamol was used as an agonist. These results suggest that beta 3-adrenoceptors exist in canine bronchial epithelium and that stimulation of this receptor subtype may enhance ciliary motility and, hence, mucociliary transport in the airway.

Inhibition of ciliary activity by phorbol esters in rabbit tracheal epithelial cells

To study the effect of protein kinase C activation on respiratory ciliary activity, we measured ciliary beat frequency (CBF) by a photoelectric technique in response to phorbol esters and cell-permeable diglyceride in cultured tracheal epithelial cells from rabbits. Phorbol 12-myristate 13-acetate (PMA) resulted in a concentration- and time-dependent inhibition of CBF (half maximum inhibitory concentration (IC50)=3×10−10 M) with the maximal decrease being 21.0±1.4% (mean±SE,p<0.001) observed at 10−6 M. L-α-dioctanoylglycerol (DiC8), another known activator of protein kinase C, likewise reduced CBF in a dose-dependent fashion. In contrast, phorbol 12,13-didecanoate, a non-tumor-promoting phorbol ester that does not stimulate protein kinase C, produced no significant changes in CBF. The decrease in CBF induced by PMA was not affected by blockade of arachidonic acid metabolism with indomethacin and nordihydroguaiaretic acid, but was antagonized by pretreatment with H-7, a specific inhibitor of protein kinase C (p<0.01). Maximal ciliary inhibition with either PMA or DiC8 was not accompanied by a decrease in intracellular concentration of cyclic AMP. These results indicate that activation of protein kinase C has a significant depressive effect on ciliary activity, and hence the airway mucociliary transport function, presumably through a regulatory pathway that is not dependent on cyclic AMP or arachidonic acid metabolites.

Adenosine Potentiates Neurally- and Histamine-Induced Contraction of Canine Airway Smooth Muscle

We studied the effect of adenosine on airway reactivity of isolated canine bronchial smooth muscle under isometric conditions in vitro. Administration of adenosine and its analogs increased the contractile responses of bronchial segments to electrical field stimulation in a dose-dependent fashion, where the rank order potency was N-ethylcarboxamideadenosine greater than adenosine greater than N-cyclohexyladenosine, but had no effect on those to exogenous acetylcholine. This potentiation was more pronounced at relatively low than at high stimulus frequencies, the maximal increase from the baseline responses being 56.3 +/- 9.6% at 1 Hz (mean +/- SE, p less than 0.01). Adenosine also increased the histamine-induced contraction causing a leftward shift of the histamine dose-response curves, an effect that was abolished in the presence of atropine. These results suggest that adenosine potentiates airway responsiveness to vagal stimulation and to histamine through the activation of prejunctional A2 receptor, probably involving the accelerated release of acetylcholine from the cholinergic nerve terminals.

Effect of Neutral Endopeptidase Inhibition on Substance-P-Induced Increase in Short-Circuit Current of Canine Cultured Tracheal Epithelium

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussings short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.

The effect of N-formyl-methionyl-leucyl-phenyl-alanine on cholinergic neurotransmission and its modulation by enkephalinase in rabbit airway smooth muscle

N-formyl-methionyl-leucyl-phenylalanine (FMLP), a synthetic analogue of bacterial chemotactic peptide, may play a role in airway hyperresponsiveness, and is cleaved by neutral endopeptidase-24.11 (enkephalinase). To determine the effect of FMLP on parasympathetic contraction of airway smooth muscle and its modulation by endogenous enkephalinase, we studied isolated rabbit tracheal ring segments under isometric conditions in vitro. FMLP did not cause muscle contraction, but it potentiated the contractile response to electrical field stimulation (EFS) in a dose-dependent fashion, with the maximal increase from the baseline response being 59.8 +/- 6.2% (mean +/- S.E.M., P less than 0.001), an effect that was abolished by t-Boc-Phe-Leu-Phe-Leu-Phe, partially inhibited by pyrilamine, but not by phentolamine or [D-Pro2,D-Trp7,9]substance P. In contrast, the contractile response to administered acetylcholine was not affected by FMLP. Pretreatment of tissues with thiorphan, an enkephalinase inhibitor, further potentiated the effect of FMLP on the EFS-induced contraction. These results suggest that FMLP facilitates cholinergic neurotransmission in rabbit airway smooth muscle probably by increasing acetylcholine release, and that this effect may be modulated by enkephalinase in the airway.

Corticotropin-releasing factor and adrenocorticotropin stimulate ciliary motility in rabbit tracheal epithelium

To assess the effects of corticotropin-releasing factor (CRF) and adrenocorticotropin (ACTH) on airway ciliary activity, we measured ciliary beat frequency (CBF) by a photoelectric method in response to these peptides in cultured rabbit tracheal explants. When cumulatively added, both CRF and ACTH increased CBF in a dose-dependent fashion. Treatment of tissues with Ca2+-free medium or nifedipine abolished the effect of CRF but not of ACTH. The CRF- and ACTH-induced ciliostimulations were not affected by indomethacin or autonomic antagonists, but were attenuated by nordihydroguaiaretic acid and by their receptor antagonists, alpha-helical CRF (9-41) and ACTH (7-38). Intracellular cyclic AMP levels were significantly increased by CRF and ACTH. These results suggest that CRF and ACTH stimulate airway ciliary motility through the activation of adenylate cyclase and lipoxygenase by binding to their specific receptors, where the effect of CRF may be triggered by Ca2+ influx.

Prophylactic effect of azelasine on sulfur dioxide-induced inhibition of ciliary motility in airway epithelium.

We studied the effects of azelastine and salbutamol on the sulfur dioxide (SO2) -induced inhibition of airway ciliary motility. Azelastine and salbutamol dose-dependently increased ciliary beat frequency (CBF) of rabbit tracheal epithelium, as detrmined by a photoelectric technique. Perfusate bubbled with 10 ppm SO2 greatly decreased CBF and intracellular cyclic AMP levels, an effect that was reversed by pretreatment of cells with azelastine and salbntamol.Thus, azelastine might protect against SO2-induced airway injury by accumulating cyclic AMP in epithelial cells.

Effect of IgE-stimulated alveolar macrophages on tracheal epithelial bioelectric properties in dogs

To investigate a possible interaction between pulmonary alveolar macrophages (AMs) and airway epithelial cells in patients with allergic conditions, we studied the effect of AMs on bioelectric properties of canine tracheal epithelium under short-circuited conditions in vitro. Mucosal addition of the supernatants from AMs stimulated with monoclonal antidinitrophenyl (DNP) IgE antibody and DNP-human serum albumin (DNP-HSA) increased short-circuit current (Isc) of cultured epithelium in a dose-dependent manner. The maximal increase from the baseline value and the EC50 were 10.2±2.0µA/cm2 (mean ± SE, p<0.01) and 3×105 AMs/ml, respectively. This effect was accompanied by the release of prostaglandin E2 and F2α from AMs. In contrast, AMs incubated with anti-DNP IgE antibody alone or DNP-HSA alone had no effect. The AM-induced increase in Isc was attenuated by diphenylamine-2-carboxylate and Cl-free medium but not by amiloride. Pretreatment of AMs with indomethacin or piroxicam inhibited the effect of AMs on epithelial Isc. These results suggest that AMs may stimulate Cl secretion across the airway mucosa through an IgE-dependent release of prostaglandins.