Simultaneous measurement of intracellular and extracellular carbonic anhydrase activity in intact muscle fibres

Abstract

The presence and properties of membrane-bound carbonic anhydrases have been difficult to establish with conventional enzymological and immunohistochemical techniques. We have therefore studied carbonic anhydrase (CA) activity in single intact crayfish muscle fibres by superfusing them alternately with a 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES)-buffered and a 5% CO2/HCO3(-)-buffered solution (pH of both solutions 7.4) while recording the intracellular pH (pHi) and extracellular surface pH (pHs) with H(+)-selective microelectrodes. In order to prevent regulation of pHi, Na+ ions were replaced with N-methyl-D-glucamine. Application of the CO2-containing solution produced a fast fall in pHi coupled with a marked (0.5-0.8 pH units) transient increase in pHs. Submicromolar concentrations of acetazolamide (AA) and benzolamide (BA) immediately blocked the pHs transients. A concentration of 8 x 10(-8) M (both compounds) reduced the response by 50%. A more prolonged application of BA and AA at concentrations of 10(-7) M and higher slowed the CO2-induced fall in pHi, which attained a rate corresponding to uncatalysed intracellular CO2 hydration at an AA concentration of 10(-4) M. The effect of BA and AA on the pHi changes developed with a time constant of 25 +/- 4 min and 7.6 +/- 1.5 min respectively, indicating that BA is less permeant than AA. CNO- ions (5 x 10(-4) M) had little effect on the CO2-induced pHs and pHi changes.(ABSTRACT TRUNCATED AT 250 WORDS)