A hallmark of human cytomegalovirus (HCMV) infection is the characteristic enlargement of the host cells (i.e., cytomegaly). Because iron (Fe) is required for cell growth and Fe chelators inhibit viral replication, we investigated the effects of HCMV infection on Fe homeostasis in MRC-5 fibroblasts. Using the metallosensitive fluorophore calcein and the Fe chelator salicylaldehyde isonicotinoyl hydrazone (SIH), the labile iron pool (LIP) in mock-infected cells was determined to be 1.04 +/- 0.05 microM. Twenty-four hours postinfection (hpi), the size of the LIP had nearly doubled. Because cytomegaly occurs between 24 and 96 hpi, access to this larger LIP could be expected to facilitate enlargement to approximately 375% of the initial cell size. The ability of Fe chelation by 100 microM SIH to limit enlargement to approximately 180% confirms that the LIP plays a major role in cytomegaly. The effect of SIH chelation on the mitochondrial membrane potential (DeltaPsi(M)) and morphology was studied using the mitochondrial voltage-sensitive dye JC-1. The mitochondria in mock-infected cells were heterogeneous with a broad distribution of DeltaPsi(M) and were threadlike. In contrast, the mitochondria of HCMV-infected cells had a more depolarized DeltaPsi(M) distributed over a narrow range and were grainlike in appearance. However, the HCMV-induced alteration in DeltaPsi(M) was not affected by SIH chelation. We conclude that the development of cytomegaly is inhibited by Fe chelation and may be facilitated by an HCMV-induced increase in the LIP.

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

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.

Intracellular organic ions exist in free solution bound to cytoplasmic proteins, partitioned within intracellular membranes, and enclosed in intracellular vesicles and organelles. The aim of this study was to develop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of diffusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluorescein (CF) was used as a model compound. It readily diffused between cytoplasm and pipette, and there was a linear relationship between concentration in the pipette and equilibrium cell fluorescence. When cells previously loaded with CF were patched, intracellular fluorescence rapidly changed in a positive or a negative direction, depending on the concentration of CF in the pipette. The null point, defined as the concentration at which cells neither gained nor lost fluorescence, described the same relationship between free concentration and total cell fluorescence as that determined by direct loading of the cells to equilibrium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 microM) for 30 min, the null point occurred at a CGamF concentration in the pipette of 0.6 microM. This value is 12 times greater than that in the bath. In conclusion, a new method is described that can measure free cytosolic concentrations of fluorescent molecules. It should prove useful in determining the intracellular location and state of transported organic ions.

previously reported that Na-K-Cl-cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV-dependent NKCC gene repression can be detected 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative immunoblotting and confocal microscopy to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is ϳ48 h, and Ͻ20% of the total expressed NKCC protein are in the plasma membrane. The remainder (ϳ80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (ϳ75%) within 24 h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h PE). Thus our results imply that, in the process of productive HCMV infection, NKCC protein continues to be synthesized, but, instead of being delivered to the plasma membrane, it is clustered in a large, detergentsoluble perinuclear structure.sodium-potassium-chloride-cotransporter; human fibroblast cell line; perinuclear accumulation