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

Abstract

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 (in [Formula: see text]-free media) was reduced in the absence of external Na+ and by treatment with bumetanide. After HCMV infection, we found that although [Cl−]iincreased 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−]iand 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.

CLIC4 mediates and is required for Ca2+-induced keratinocyte differentiation

“…Nuclear chloride content was measured semi-quantitatively and qualitatively detected by confocal microscopy using the chloride-ion-sensitive fluorescent dye N-(6-methoxyquinolyle) acetoethyl ester (MQAE; Molecular Probes/Invitrogen) based on the methods described by the manufacturer and Maglova et al (Maglova et al, 1998a;Maglova et al, 1998b). The chloride channel inhibitor NPPB was purchased from BioMol (Plymouth Meeting, PA).…”

Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

“…The drastically decreased NKCC function was paralleled, and could be explained, by a severe reduction (approximately ninefold) of NKCC protein expression in the membrane subfraction (determined 72 h PE; Ref. 41).…”

“…Therefore, it is not surprising that HCMV infection is characterized by significant effects on a variety of membrane ion transporters that mediate Na ϩ transmembrane movements. Examples include the sodium pump (3,41,46) and the Na ϩ /H ϩ exchanger (NHE) (9), as well as the Cl Ϫ /HCO 3 Ϫ exchanger, which works in concert with the NHE to mediate net uptake of inorganic osmolytes promoting cell volume increases (37). Fons et al (18) reported that treatment of HCMV-infected cells with amiloride (an inhibitor of NHE) inhibited HCMV replication by almost two orders of magnitude.…”

“…41). The bumetanide and Na ϩ sensitivities of intracellular [Cl Ϫ ], as well as net Cl Ϫ uptake and loss, were reduced by approximately threefold within 24 h postexposure (PE) to HCMV and abolished by 72 h PE.…”

Dysfunction in Respiration and Osmotic Regulation of Larval Japanese Flounder Affected by Viral Epidermal Hyperplasia

“…5 and 6), suggesting poorer osmoregulatory ability for these fish. In addition, suppression of the expression of NKCC in chloride cells may be involved in the reduced osmoregulatory function, as is reported by Maglova et al (1998) where MRC-5 cell line (human lung fibroblast) was infected with human cytomegalovirus (HCMV, a herpesvirus). Thus, it is reasonable that the survival rate of FHV-infected fish was higher in seawater with lower osmolarities that are closer to the physiological osmolarity in the fish body.…”