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

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.