ABSTRACT
We investigated regulatory mechanisms of Cl(-) secretion playing an essential role in the maintenance of surface fluid in human airway epithelial Calu-3 cells. The present study reports that quercetin (a flavonoid) stimulated bumetanide-sensitive Cl(-) secretion with reduction of apical Cl(-) conductance, suggesting that quercetin stimulates Cl(-) secretion by activating an entry step of Cl(-) across the basolateral membrane through Na(+)/K(+)/2Cl(-) cotransporter (NKCC1). To clarify the mechanism stimulating NKCC1 by quercetin, we verified involvement of protein kinase (PK)A, PKC, protein tyrosine kinase (PTK), and cytosolic Ca(2+)-dependent pathways. A PKA inhibitor (PKI-14-22 amide), a PKC inhibitor (Gö 6983) or a Ca(2+) chelating agent did not affect the quercetin-stimulated Cl(-) secretion. On the other hand, a PTK inhibitor (AG18) significantly diminished the stimulatory action of quercetin on Cl(-) secretion without inhibitory effects on apical Cl(-) conductance, suggesting that a PTK-mediated pathway is involved in the stimulatory action of quercetin. The quercetin action on Cl(-) secretion was suppressed with brefeldin A (BFA, an inhibitor of vesicular transport from ER to Golgi), and the BFA-sensitive Cl(-) secretion was not observed in the presence of an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478), suggesting that quercetin stimulates Cl(-) secretion by causing the EGFR kinase-mediated translocation of NKCC1 or an NKC1-activating factor to the basolateral membrane in human airway epithelial Calu-3 cells. However, the surface density of NKCC1 was not increased by quercetin, but quercetin elevated the activity of NKCC1. These observations indicate that quercetin stimulates Cl(-) secretion by activating NKCC1 via translocation of an NKCC1-activating factor through an EGFR kinase-dependent pathway.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Quercetin/pharmacology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Sodium-Potassium-Chloride Symporters/metabolism , Brefeldin A/pharmacology , Bumetanide/pharmacology , Cell Line , Chlorides/metabolism , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Humans , Ion Transport/drug effects , Quinazolines , Respiratory Mucosa/cytology , Signal Transduction/drug effects , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Solute Carrier Family 12, Member 2 , Tyrphostins/pharmacology , Valinomycin/pharmacologySubject(s)
Antineoplastic Agents/therapeutic use , Hemangiosarcoma/drug therapy , Interleukin-2/therapeutic use , Nose Neoplasms/drug therapy , Follow-Up Studies , Hemangiosarcoma/diagnosis , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Nose Neoplasms/diagnosis , Recombinant Proteins/therapeutic use , Tomography, X-Ray ComputedABSTRACT
In order to complete TDM manual for pirmenol in Sapporo Medical Center NTT East, we developed HPLC method and pretreatment procedure for pirmenol samples obtained from patients. Serum (250 microliters) was alkalinized and pirmenol was extracted into n-hexane, and then the drug was again extracted into an acidic solvent, 0.044 M KH2PO4 (pH 2.6) including 0.5% triethylamine. The aqueous extract was used for quantitative determination of the drug by HPLC. The mobile phase consisted of the above acidic solvent-acetonitrile (5:1, v/v) was delivered at 45 degrees C with a flow rate of 1 ml/min through a 4.6 mm x 25 cm ODS-3, a reversed-phase column. Detection of pirmenol and the internal standard (disopyramide) was achieved at 263 nm. Pirmenol and disopyramide was eluted at 5 and 11 min, respectively. Assay limit (25 ng/ml) and accuracy of the analytical method were satisfactory for TDM of pirmenol. During the HPLC analysis of patient samples, no substances that interfered with pirmenol detection were found. It was shown that 1) hemolysis did not affect pirmenol assay at all, 2) pirmenol was stable in the blood samples for at least 24 h even if they were stood at room temperature, and 3) pirmenol was stable for at least 3 days in frozen serum but there significant decrease was observed in pirmenol concentration after 7 days.
