title: Na+/K+-ATPase α1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role in the barrier integrity.
abstract: BACKGROUND
  The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+)/K(+)-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.
  
  
  RESULTS
  Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.
  
  
  CONCLUSIONS
  The results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier.
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, Oregon Hearing Research Center, Oregon Health & Science University, Portland, Oregon, United States of America.
authors.forename: Yue
authors.initials: Y
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Dai
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Wilson
authors.forename: Irina
authors.initials: I
authors.lastname: Omelchenko
authors.forename: John E
authors.initials: JE
authors.lastname: Klimek
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Wilmarth
authors.forename: Larry L
authors.initials: LL
authors.lastname: David
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Nuttall
authors.forename: Peter G
authors.initials: PG
authors.lastname: Gillespie
authors.forename: Xiaorui
authors.initials: X
authors.lastname: Shi
mesh_terms: D000818:Animals; D002196:Capillaries; D030562:Databases, Protein; D007758:Ear, Inner / Q000098:blood supply / Q000201:enzymology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D062793:Occludin; D011493:Protein Kinase C / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism / Q000502:physiology*
chemical_list: D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; C081975:protein kinase C eta; D011493:Protein Kinase C; C497344:Atp1a1 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase