LOW PH ENHANCES EXPRESSION OF CARBONIC-ANHYDRASE-II BY CULTURED RAT INNER MEDULLARY COLLECTING DUCT CELLS

Carbonic anhydrase (CA) facilitates the secretion of protons from rena l epithelia by catalyzing the buffering of hydroxyl ions by CO2. We ha ve previously found that inner medullary collecting duct (IMCD) cells cultured from rat kidney secrete protons and express CA II. Incubation of IMCD cells in acidic medium for 48 h has been shown to stimulate t he secretion of protons by a protein synthesis-dependent process. To e stablish whether CA II might be involved in this process, IMCD cells w ere exposed to incubation media supplemented with 10(-7) M deoxycortic osterone acetate, pH 7.0 (acid) or pH 7.7 (control) for 48 h, and CA I I mRNA and protein were quantitated. Part of the CA II cDNA was obtain ed by reverse transcription of total RNA from rat kidney followed by a mplification using oligonucleotide primers derived from conserved area s in the coding regions of human, mouse, and chick CA II cDNAs in a po lymerase chain reaction. By Northern analysis, steady-state levels of CA II mRNA from acid-incubated cells showed an increase of 80% compare d with controls and 70% when expressed relative to a housekeeping mRNA , p-actin. Western blot analysis using a human antibody to CA II showe d an approximate doubling of CA II protein after acid incubation. By i mmunofluorescence microscopy, the domes of acid-incubated IMCD cells c ontained considerably more CA II-stained cells than found in control c ultures. Thus incubation of IMCD cells in acid medium stimulates the e xpression of CA II mRNA and protein. This upregulation of CA II may be part of the adaptive response to low pH and mag facilitate the increa sed rate of proton pumping observed under these conditions.