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 (125)I-rProtein A as a tracer. The mean (125)I 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 10(7) CA-II molecules were quantified for 1 microg 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..

Fig. (1)

SDS-PAGE and Densitometry analysis of carbonic anhydrase II. The densitometric profile in upper figure shows the standard peak optical density and distance from the start point, which represents different molecular weight protein and corresponds to SDS-PAGE detected proteins. The standard CA-II protein is at the position 31.0 kDa band. Lower figure shows the peaks and distances of immunoreactive proteins extracted from carotid body obtained by SDS-PAGE. An immunoreactive protein peak showed at position 31 kDa confirms the presence of CA II in rat carotid body. Only the molecular weight region between 97.4 and 14.4 kDa is shown.

Fig. (2)

Western blot analysis of carbonic anhydrase II. The digital image of immunoreactive band from rat CB extract is at position of 31 kDa, which represents the CA-II, as evidenced by the standard bovine carbonic anhydrase-II marker. CB: carotid body; M: standard control marker.

Fig. (3)

Correlation between ¹²⁵I radioactivity and amount of standard carbonic anhydrase. The figure shows a positive linear relationship between ¹²⁵I radioactivity reading (cpm) and carbonic anhydrase weight (µg). Data are represented as mean value of three independent curves.