Identification and Measurement of Carbonic Anhydrase-II Molecule Numbers in the Rat Carotid Body



Guglielmo Di Tano1, Claudia Petrarca1, Gerardo Bosco1, Battista Pasquale2, Zhongjin Yang*, 3, Luca Morelli1, Renato Barbacane4, Bruno Loffredo1
1 Department of Basic and Applied Medical Sciences, G. d’Annunzio University of Chieti-Pescara, School of Medicine, Chieti, Italy
2 Motor sciences, University of Chieti, Chieti-Pescara, Italy
3 Institute for Human Performance, Upstate Medical University, Syracuse, NY, USA
4 Immunology Division, G. d’Annunzio University of Chieti-Pescara, School of Medicine, Chieti, Italy


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© Di Tano et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Department of Anesthesiology, SUNY Upstate Medical University, Syracuse, New York, 13210. USA;; E-mail: yangz@upstate.edu


Abstract

Carbonic anhydrase (CA) in the carotid body (CB) plays an important role in the maintenance of blood PO2 and PCO2/pH homeostasis by regulating ventilation. It has been observed that the activity of CA in the rabbit CB is stronger under hypoxic conditions than under normoxic and hyperoxic conditions. In conditions of chronic hypoxia, the volume of the CB increases significantly because the number of type I and II cells increases. So far, the number of CA molecules in the CB has not been assessed. We develop a technique to quantify the number of CA molecules in the CB. The CBs were dissected out from 8 rats, immediately frozen with liquid nitrogen, pulverized and centrifuged. The proteins extracted from CB tissue were heat-denatured and separated by electrophoresis on a 12.5% denatured-polyacrylamide gel (SDSPAGE); a 31 kDa protein band was determined which reacted with a rabbit polyclonal antibody specific for rat CA-II in Western blot analysis. The immunoreactive 31 kDa CA-II protein was detected and quantified by laser scanner densitometry using 125I-rProtein A as a tracer. The mean 125I radioactivity emitted by the antibody bound CA-II was 31277 cpm. This value corresponds to 4.57 ng CA-II. When compared with a rat CA-II calibration curve, an average of number of 3.54 x 107 CA-II molecules were quantified for 1 µg of whole CB tissue. This is a sensitive and accurate radioimmunoassay technique and may be useful in future studies on the role of CA-II in different pathophysiologic conditions.

Keywords: Carbonic anhydrase, Carotid body, Phosphor imaging, SDS-PAGE, Western blot..