Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV) infection on the Na+-K+-Cl- cotransporter (NKCC) in a human fibroblast cell line. Using the Cl(-)sensitive dye MQAE, we showed that the mock-infected MRC-5 cells express a functional NKCC. 1) Intracellular Cl- concentration ([Cl-](i)) was significantly reduced from 53.4 +/- 3.4 mM to 35.1 +/- 3.6 mM following bumetanide treatment. 2) Net Cl- efflux caused by replacement of external Cl- with gluconate was bumetanide sensitive. 3)In Cl--depleted mock-infected cells, the Cl- reuptake rate tin HCO3--free media) was reduced in the absence of external Na+ and by treatment with bumetanide. After HCMV infection, we found that although [Cl-](i) increased progressively [24 h postexposure (PE), 65.2 +/- 4.5 mM; 72 h PE, 80.4 +/- 5.0 mM], the bumetanide and Na+ sensitivities of[Cl-]i and net Cl- uptake and loss were reduced by 24 h PE and abolished by 72 h PE. Western blots using the NKCC-specific monoclonal antibody T4 showed an approximately ninefold decrease in the amount of NKCC protein after 72 h of infection. Thus HCMV infection resulted in the abolition of NKCC function coincident with the severe reduction in the amount of NKCC protein expressed.