REGULATION OF PHAGOSOMAL ACIDIFICATION - DIFFERENTIAL TARGETING OF NA+ H+ EXCHANGERS, NA+/K+-ATPASES, AND VACUOLAR-TYPE H+-ATPASES/

Vacuolar type (V) ATPases are thought to be the main determinant of ph agosomal acidification, In phagosomes containing mycobacteria, which o stensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral, This prediction was test ed by microfluorescence ratio imaging using macrophages from mice susc eptible to mycobacterial infection, Although less acidic than their co unterparts containing dead bacteria, phagosomes containing live Mycoba cteria bovis were nearly 1 pH unit more acidic than the cytosol, sugge sting the existence of alternate H+ transport mechanisms, We there for e investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification, Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the pred ominant isoform of the exchanger in macrophages. Fractionation reveale d that NHE1 is incorporated into the phagosomal membrane, and measurem ents of pH indicated that it is functional in this location, Neverthel ess, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidi nobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1, Thi s may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid o f Na+/K+ pumps, Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced qua ntities in the phagosomal membrane, Alternatively, acid equivalents ac cumulated in endosomes by V-ATPases may be delivered to the mycobacter ial phagosome by carrier vesicles devoid of ATPases.