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 (in HCO-3-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.

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts.

The effects of human cytomegalovirus (HCMV) infection on Cl-/HCO-3 exchanger activity in human lung fibroblasts (MRC-5 cells) were studied using fluorescent, ion-sensitive dyes. The intracellular pH (pHi) of mock- and HCMV-infected cells bathed in a solution containing 5% CO2-25 mM HCO-3 were nearly the same. However, replacement of external Cl- with gluconate caused an H2DIDS-inhibitable (100 microM) increase in the pHi of HCMV-infected cells but not in mock-infected cells. Continuous exposure to hyperosmotic external media containing CO2/HCO-3 caused the pHi of both cell types to increase. The pHi remained elevated in mock-infected cells. However, in HCMV-infected cells, the pHi peaked and then recovered toward control values. This pHi recovery phase was completely blocked by 100 microM H2DIDS. In the presence of CO2/HCO-3, there was an H2DIDS-sensitive component of net Cl- efflux (external Cl- was substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl- (in the nominal absence of CO2/HCO-3), the H2DIDS-sensitive net Cl- efflux was much greater from HCMV- than from mock-infected cells. In mock-infected cells, H2DIDS-sensitive, net Cl- efflux decreased as pHi increased, whereas for HCMV-infected cells, efflux increased as pHi increased. All these results are consistent with an HCMV-induced enhancement of Cl-/HCO-3 exchanger activity.

Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+ exchange in human fibroblasts.

Infection with human cytomegalovirus (HCMV) causes an enlargement (cytomegaly) of human fibroblasts (MRC-5). As a first step toward determining whether solute uptake, mediated in part by Na+/H+ exchange, is responsible for the development of cytomegaly, we studied the effects of HCMV infection on intracellular pH (pHi) regulation (nominal CO2/ HCO3- concn = 0) by comparing cytomegalic cells with mock-infected cells. Seventy-two hours after HCMV infection of MRC-5 cells we observed the following changes relative to mock-infected cells: resting pHi is 0.1-0.2 pH unit more alkaline; the intrinsic buffering power of the cytoplasm was reduced by approximately 40-50%; acid-loading H(+)-equivalent fluxes were reduced; and there were alterations of Na+/H+ exchanger (NHE) properties, including an alkaline shift of the pHi dependence of activity, a reduction of the apparent affinity for extracellular Na+, and an increase of the apparent maximum velocity and a large increase in stimulation by a hyperosmotic challenge. These results indicate that HCMV infection exerts a profound effect on functional properties of the NHE, on acid-loading mechanisms, and on intrinsic cellular buffering power. These effects are consistent with a role for the NHE in the development of cytomegaly.

Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe.

A non-invasive microspectrofluorimetric technique was used to investigate experimentally induced changes in cell water volume in single N1E-115 murine neuroblastoma cells, using calcein, a derivative of fluorescein, as a marker of the intracellular water compartment. The osmotic behavior of N1E-115 cells exposed to media of various osmolalities was studied. Exposure to hyperosmotic (up to +28%) or hyposmotic (up to -17%) solutions produced reversible decreases and increases in cell water volume, respectively, which agreed with near-osmometric behavior. Increases in [Ca2+]i produced by exposing the cells to the ionophore ionomycin (1 microM) in isosmotic medium, resulted in a gradual decrease in cell water volume. Cells shrank to 40 +/- 7% (n = 7) below their initial water volume at an initial rate of -1.2 +/- 0.2%/min. It is concluded that N1E-115 cells are endowed with Ca2+-sensitive mechanisms for volume control, which can produce cell shrinkage when activated under isosmotic conditions. Because the technique used for measuring cell water volume changes is new, we describe it in detail. It is based on the principle that relative cell water volume in single cells can be measured by introducing an impermeant probe into cells and measuring its changes in concentration. If the intracellular content of the probe is constant, changes in its concentration reflect changes in cell water volume. Calcein was used as the probe because its fluorescence intensity is directly proportional to its concentration and independent of changes in the concentration of native intracellular ions within the physiological range. Because calcein is two to three times more fluorescent that other fluorophores such as 2,7,-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein or Fura-2, and it is used at its peak excitation and emission wavelengths, it has a better signal to noise ratio and baseline stability than the other dyes. Calcein can also be esterified allowing for cell loading and because of the possibility of reducing the intensity of the excitation light, measurements can be performed producing minimal photodynamic damage. The technique allows for measurements of cell water volume changes of < 5% and it can be applied to single cells which can be grown or affixed to a rigid substratum, e.g., a coverslip.

Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells.

To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes.

Resistive properties of the epithelial membranes of the urinary bladder of the toad, Bufo marinus, determined using the fluorescent dye, RH160.

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range +/- 200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 mumol.l-1), reduced by bretylium (5 mmol.l-1) and abolished in the presence of nystatin (730 U.ml-1). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 k omega.microF, and the basolateral membrane resistance was 1.5 k omega.microF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.

Role of intracellular Ca2+ in modulation of tight junction resistance in A6 cells.

The role of intracellular Ca2+ in the development and maintenance of epithelial tight junctional integrity is poorly understood. We assessed tight junctional resistance (Rj) in confluent monolayers of A6 cells that were treated with mucosal amiloride such that the transepithelial resistance (Rt) reflects Rj. Solution Ca2+ concentration [Ca2+] was reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) addition to the bathing solutions. Reduction of mucosal [Ca2+] to 1 microM or reduction of serosal Ca2+ to 100 microM did not significantly alter Rt. However, a further decrease of serosal Ca2+ to 40 microM caused the resistance to fall to < 12% of the control value. Following restoration of serosal [Ca2+], Rt increased to a new steady-state value within approximately 15 min. The magnitude of recovery of Rt was inversely correlated with the length of time the epithelium was exposed to low serosal [Ca2+]. To further test the effects of asymmetric Ca2+ removal, the serosal [Ca2+] was chelated using EGTA to reduce Rt. When the Ca2+ ionophore A-23187 was subsequently added to the mucosal solution, Rt increased from 20% to 60% of the control level. In addition, cells were loaded with the fluorescent Ca2+ indicator, Calcium Green, and the temporal relationship between changes in Rt and intracellular Ca2+ was determined. Following removal of serosal Ca2+, cell Ca2+ decreased, followed by a decrease in Rt. In contrast, returning Ca2+ to the serosal bathing solution resulted in a parallel increase of both Rt and cell [Ca2+]. These data strongly suggest that changes in intracellular [Ca2+] play an important role in the regulation of Rj.

A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope.

In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na+ transport rate (measured as short-circuit current, ISC) increased. The increase in cell height preceded changes in ISC by several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na+ transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.

Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.

Solution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (I(sc)). I(sc) for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25 +/- 2, 9 +/- 2, 3 +/- 0.4, and 0.6 +/- 0.5 microA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity: mucosal changes did not significantly affect I(sc). The changes in I(sc) were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Occult proximal deep vein thrombosis: its prevalence among patients admitted to a rehabilitation hospital.

This study was designed first to determine the prevalence of occult proximal deep vein thrombosis (DVT) in stroke patients admitted to rehabilitation hospital using the technique of impedance plethysmography (IPG), and second, to identify clinical findings which may be indicators of an increased risk for the development of proximal DVT. Impedance plethysmography was performed on 105 consecutive stroke patients within one week of admission to our hospital. It was found that 34 out of 100 patients with adequate studies had abnormal IPG, two out of the 34 had known DVT, leaving 32 out of 98 with undiagnosed DVT (19 on the paretic side alone, nine bilateral, and four on the nonparetic side). Using logistic regression analysis, it was determined that profound weakness, male gender, interval between the stroke and IPG, edema, and leg hyperpigmentation were independently associated with positive IPG. Since IPG has a high positive predictive value for proximal DVT, one must assume that most of our patients with positive IPG have proximal DVT. Routine screening of stroke patients for DVT seems indicated and probably should include noninvasive venous studies such as serial IPG. The most efficient screening protocol needs to be determined.

The autologous mixed lymphocyte reaction is decreased in Freund's adjuvant-injected rats of arthritis-susceptible and -insusceptible strains.

We examined the T-non-T cell autologous mixed lymphocyte reaction (AMLR) of spleen cells from rats with arthritis induced by Freund's complete adjuvant in an effort to establish an animal model for the study of the relationship between the AMLR and autoimmune disease. We found that the splenic T-non-T AMLR was markedly decreased in rats with adjuvant-induced arthritis and that this decrease was mediated by suppressor cells within the nylon-wool-adherent stimulator cell population. However, we also found a similar decrease in the AMLR of arthritis-resistant Fisher 344 rats that received Freund's complete adjuvant but did not develop arthritis. Control animals with local inflammation induced by turpentine, a non-arthritogenic inflammatory substance, had normal AMLR, whereas other controls given Freund's incomplete adjuvant, also a non-arthritogenic substance, had a modest responder cell-mediated decrease in AMLR. These studies help to clarify the relationship between the decreased AMLR and the pathogenesis of adjuvant-induced arthritis by demonstrating that: 1) the acute-phase inflammatory response does not reduce the AMLR; and 2) the decreased AMLR can occur in the absence of overt autoimmune disease. This latter observation calls into question the proposed pathogenetic relationship between the AMLR and autoimmune disease states.

Immunogenetic studies of juvenile dermatomyositis. III. Study of antibody to organ-specific and nuclear antigens.

Ninety children with definite juvenile dermatomyositis (JDMS), who had been HLA typed, were tested for the presence of tissue or organ-specific antibodies. Sixty had active disease at the time of study. The mean disease duration was 4 years, and 30 had soft tissue calcifications. The following autoantibodies were sought: thyroid, gastric parietal cells, smooth muscle, striated muscle, microsomes, mitochondria, DNA, extractable nuclear antigen, Sm, PM-1, antinuclear antibody (ANA), and rheumatoid factor. Only the ANA and PM-1 were more frequent in patients than in controls (P less than 0.0002 and P less than 0.001, respectively). Higher levels of immune complexes (P less than 0.01) were found in sera from patients with JDMS than in sera from controls and were correlated with the presence of ANA in patients (P less than 0.01). Soft tissue calcification was not associated with any autoantibody or HLA antigen, but with disease duration and activity (P less than 0.001 and P less than 0.05, respectively). There was no association between the occurrence of any autoantibody and the presence of HLA-B8 or DR3 among the white patients with JDMS. The frequency of autoantibodies in 43 full siblings of children with JDMS was not increased. We conclude that children with JDMS, with or without HLA-B8/DR3, do not show evidence of a generalized nonspecific antibody response to tissue antigens. The significance of the increased antibody to nuclear antigens ANA and PM-1 remains to be determined.

Nailfold capillary abnormalities in childhood rheumatic diseases.

The nailfold capillary patterns of 84 patients with a variety of childhood rheumatic diseases and 34 normal control subjects were observed. Distinctive morphologic abnormalities with capillary dilation and dropout of surrounding structures were noted in two groups: patients with childhood dermatomyositis and with scleroderma (P less than 0.001). Among those with scleroderma, capillary abnormalities were found in all nine patients with systemic disease and in none of 10 patients with cutaneous disease only (Fisher's exact P less than 0.001). Of 25 patients with dermatomyositis for whom muscle biopsies were available for analysis, abnormal nailfold capillary pattern was found with highest prevalence in patients with two or more specific vascular lesions noted on biopsy (Fisher's exact P = 0.041). Nailfold capillary abnormalities are present in distinct populations of childhood rheumatic diseases, reflect the underlying vasculopathy of childhood dermatomyositis, and may be of diagnostic value in distinguishing localized from systemic scleroderma.

Immunogenetic studies of juvenile dermatomyositis. HLA antigens in patients and their families.

Typing for HLA-A and -B antigens was performed on 87 children with definite juvenile dermatomyositis (JDMS). A significantly increased frequency of HLA-B8 (estimated relative risk = 2.8, Pc less than 0.01) was observed among White patients, but not among Blacks or Latin Americans with JDMS. No abnormality of HLA haplotype segregation was observed among 38 healthy siblings of the JDMS probands.

Nailfold capillary abnormalities and clinical outcome in childhood dermatomyositis.

The nailfold capillary pattern was observed in a population of patients with childhood dermatomyositis. Distinctive nailfold capillary loop abnormalities were found in 11 of 19 childhood dermatomyositis patients and in none of 2 control populations (P less than 0.001). By a retrospective analysis of the childhood dermatomyositis patients, we found that the presence of nailfold capillary abnormalities correlates with more severe forms of the disease (ulcerative and chronic types), as opposed to limited type of disease. These changes occurred independently of disease activity or of cutaneous abnormalities.