title: cAMP-stimulated Cl- secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium.
abstract: BACKGROUND
  The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.
  
  
  RESULTS
  Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2'-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/- mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR-/- mice had no responses. Nonetheless, CFTR-/- mice showed no difference from CFTR+/- mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined.
  
  
  CONCLUSIONS
  These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.
authors.affiliation: Dept, Anatomy & Physiology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Satyanarayana R
authors.initials: SR
authors.lastname: Pondugula
authors.forename: Suresh B
authors.initials: SB
authors.lastname: Kampalli
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.forename: Robert C
authors.initials: RC
authors.lastname: De Lisle
authors.forename: Nithya N
authors.initials: NN
authors.lastname: Raveendran
authors.forename: Donald G
authors.initials: DG
authors.lastname: Harbidge
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Marcus
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology*; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects* / Q000378:metabolism; D005938:Glucocorticoids / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D012665:Semicircular Canals; D012964:Sodium / Q000378:metabolism
chemical_list: D002712:Chlorides; D005938:Glucocorticoids; D010726:Phosphodiesterase Inhibitors; D011965:Receptors, Glucocorticoid; D002034:Bumetanide; D005576:Colforsin; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D011188:Potassium
references.citation: J Clin Invest. 2002 Dec;110(11):1651-8
references.pmid: 12464670
references.citation: J Pharmacol Exp Ther. 2004 Jul;310(1):108-15
references.pmid: 14996950
references.citation: Biochim Biophys Acta. 2004 Nov 17;1667(1):7-14
references.pmid: 15533301
references.citation: Am J Hypertens. 1990 Dec;3(12 Pt 1):939-42
references.pmid: 2081015
references.citation: Hypertension. 2003 May;41(5):1131-5
references.pmid: 12668585
references.citation: J Membr Biol. 2000 Jun 1;175(3):191-202
references.pmid: 10833529
references.citation: Neuropharmacology. 2004 Aug;47(2):253-62
references.pmid: 15223304
references.citation: Biochem J. 1996 Sep 15;318 ( Pt 3):1057-63
references.pmid: 8836156
references.citation: Nature. 2003 Aug 21;424(6951):938-42
references.pmid: 12931188
references.citation: Acta Otolaryngol Suppl. 1993;501:76-9
references.pmid: 8447231
references.citation: J Smooth Muscle Res. 2005 Feb;41(1):1-21
references.pmid: 15855736
references.citation: J Membr Biol. 2005 Sep;207(1):11-22
references.pmid: 16463139
references.citation: J Clin Invest. 1981 Mar;67(3):770-8
references.pmid: 7204555
references.citation: Physiol Rev. 2012 Oct;92(4):1577-617
references.pmid: 23073627
references.citation: Annu Rev Physiol. 1997;59:365-93
references.pmid: 9074769
references.citation: Invest Ophthalmol Vis Sci. 1997 May;38(6):1229-40
references.pmid: 9152243
references.citation: Cell Physiol Biochem. 2001;11(6):295-310
references.pmid: 11832655
references.citation: BMC Med. 2004 Aug 20;2:30
references.pmid: 15320950
references.citation: Am J Physiol Cell Physiol. 2002 Apr;282(4):C817-23
references.pmid: 11880270
references.citation: Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L217-30
references.pmid: 11404265
references.citation: Pflugers Arch. 1986;407 Suppl 2:S128-41
references.pmid: 2434915
references.citation: Audiol Neurootol. 2002 Mar-Apr;7(2):100-6
references.pmid: 12006737
references.citation: Jpn J Pharmacol. 2000 Nov;84(3):244-51
references.pmid: 11138724
references.citation: J Biol Chem. 2000 Sep 29;275(39):30326-34
references.pmid: 10913127
references.citation: Physiology (Bethesda). 2006 Oct;21:326-35
references.pmid: 16990453
references.citation: Trends Pharmacol Sci. 2012 Feb;33(2):64-78
references.pmid: 22100304
references.citation: J Membr Biol. 1997 Mar 1;156(1):45-52
references.pmid: 9070463
references.citation: Neuroscience. 2003;119(1):3-8
references.pmid: 12763062
references.citation: Hear Res. 1997 Dec;114(1-2):107-16
references.pmid: 9447925
references.citation: Mol Pharmacol. 1988 Jun;33(6):696-701
references.pmid: 3380083
references.citation: Am J Physiol. 1997 Nov;273(5):C1516-25
references.pmid: 9374636
references.citation: Am J Physiol. 1996 Feb;270(2 Pt 1):G259-67
references.pmid: 8779967
references.citation: J Membr Biol. 2002 Jan 15;185(2):137-44
references.pmid: 11891572
references.citation: Hear Res. 1994 Jul;78(1):58-64
references.pmid: 7961178
references.citation: Am J Physiol. 1996 Aug;271(2 Pt 1):C486-94
references.pmid: 8769987
references.citation: Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G899-906
references.pmid: 11557509
references.citation: J Membr Biol. 1997 Mar 1;156(1):25-35
references.pmid: 9070461
references.citation: Am J Respir Cell Mol Biol. 1995 Sep;13(3):307-13
references.pmid: 7544595
references.citation: Annu Rev Pharmacol Toxicol. 1996;36:461-80
references.pmid: 8725398
references.citation: Am J Physiol Cell Physiol. 2009 Sep;297(3):C688-98
references.pmid: 19535515
references.citation: Am J Physiol Cell Physiol. 2002 Dec;283(6):C1752-60
references.pmid: 12388054
references.citation: Am J Physiol Renal Physiol. 2004 Jun;286(6):F1127-35
references.pmid: 14996671
references.citation: Physiol Rev. 1999 Jan;79(1 Suppl):S109-44
references.pmid: 9922378
references.citation: J Clin Invest. 1983 Sep;72(3):997-1004
references.pmid: 6309913
references.citation: Physiol Genomics. 2006 Jan 12;24(2):114-23
references.pmid: 16263802
references.citation: Comp Biochem Physiol A Mol Integr Physiol. 2012 Jan;161(1):53-60
references.pmid: 21964154
references.citation: Am J Physiol Gastrointest Liver Physiol. 2010 Jul;299(1):G81-95
references.pmid: 20413718
references.citation: Curr Eye Res. 1989 Jan;8(1):85-90
references.pmid: 2565197
references.citation: Clin Exp Pharmacol Physiol. 2000 Nov;27(11):892-5
references.pmid: 11071305
references.citation: Cell Physiol Biochem. 2009;23(4-6):407-14
references.pmid: 19471108
references.citation: Eur J Pediatr. 1993 Mar;152(3):265-6
references.pmid: 8444259
references.citation: J Membr Biol. 1999 Jul 1;170(1):67-77
references.pmid: 10398761
references.citation: Am J Physiol Cell Physiol. 2008 Mar;294(3):C820-32
references.pmid: 18184876
references.citation: Arch Otolaryngol. 1979 Jun;105(6):338-42
references.pmid: 454286
references.citation: Biochem Biophys Res Commun. 2005 Jun 17;331(4):1353-7
references.pmid: 15883024
references.citation: J Neurochem. 1994 Oct;63(4):1271-6
references.pmid: 7931278
references.citation: J Pharmacol Exp Ther. 2005 Aug;314(2):846-54
references.pmid: 15901792
references.citation: Int J Mol Sci. 2011;12(12):9125-37
references.pmid: 22272123
references.citation: Hear Res. 1987;30(1):55-64
references.pmid: 3680054
references.citation: Am J Physiol Cell Physiol. 2009 Mar;296(3):C544-57
references.pmid: 19144862
references.citation: Biochem J. 2003 Nov 1;375(Pt 3):517-29
references.pmid: 12940771
references.citation: BMC Res Notes. 2010 Apr 14;3:100
references.pmid: 20398257
references.citation: Pflugers Arch. 2001;443 Suppl 1:S85-91
references.pmid: 11845310