Altered Na+/H+ exchanger isoform 1 activity in immortalized lymphoblasts from women with pre-eclampsia: evidence for an intermediate phenotype.

Previous studies have demonstrated a raised Na(+) content in leucocytes isolated from women with pre-eclampsia. Increased Na(+)/H(+) exchanger activity is one membrane transport abnormality that may contribute to this phenomenon and may be implicated in the abnormal volume homoeostasis and hypertension associated with the disease. Increased Na(+)/H(+) exchanger activity has been documented in nucleated white blood cells from both pre-eclamptic and post-partum pre-eclamptic women, and may suggest the importance of genetic influences on exchanger activity. In the present study, we used lymphoblasts from women with pre-eclampsia and from age- and gestation-matched normotensive pregnant controls to determine Na(+)/H(+) exchanger activity and intracellular resting pH using fluorimetry and the pH-sensitive dye BCECF-AM [bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester]. Determination of Na(+)/H(+) exchanger protein abundance was performed by Western blotting. Intracellular pH was not significantly different in cells from pre-eclamptic women compared with those from normotensive controls. Na(+)/H(+) exchanger activity was measured when the intracellular pH was clamped at 6.0, and was found to be significantly higher in cells from pre-eclamptic women (20.77+/-0.92 mmol x min(-1) x l(-1)) compared with those from normotensive controls (15.22+/-0.92 mmol x min(-1) x l(-1); P =0.001). Na(+)/H(+) exchanger protein abundance was established to be similar in the two subject groups, suggesting that the turnover number for the Na(+)/H(+) exchanger is increased in the women with pre-eclampsia. These changes in Na(+)/H(+) exchanger activity indicate the importance of genetic factors in determining this particular phenotype, since in this cell culture model of pre-eclampsia it is likely that environmental or hormonal influences present in vivo would have declined. Overactivity of the Na(+)/H(+) exchanger may contribute to the raised intracellular Na(+) concentration reported previously in white blood cells from women with pre-eclampsia.

Potassium and activity of the sodium hydrogen exchanger isoform 1 in vascular smooth muscle of hypertensive rats.

Low potassium intake is inversely associated with blood pressure. In vitro, the proliferation of vascular smooth muscle cells (VSMCs) shows an inverse correlation with [K(+)]. In hypertension, many studies have established that the ubiquitous Na(+)/H(+) exchanger isoform 1 (NHE-1) exhibits increased activity and is permissive for cell proliferation. Changes in extracellular [K(+)] lead to altered intracellular Na(+) content, which could affect NHE activity and NHE-1 protein expression. We therefore investigated the effects of altering extracellular [K(+)] on NHE activity and NHE-1 expression in cultured VSMCs of both the spontaneously hypertensive rat (SHR) and its normotensive Wistar-Kyoto counterpart (WKY). Culture of SHR VSMCs for 48 hours in media containing 2, 4, 6, and 8 mmol. L(-1) [K(+)] led to activation of NHE-1 in the low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 34.3 +/- 1.7, 29.5 +/- 1.1, 27.7 +/- 1.4, and 26.1 +/- 2.1 mmol. L(-1) min(-1), P <.006 by analysis of variance [ANOVA]). This was not associated with any significant changes in intracellular pH. By contrast, WKY VSMCs did not exhibit any significant activation of NHE-1 in low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 24.3 +/- 2.9, 22.3 +/- 1.7, 19.0 +/- 1.8, and 18.6 +/- 1.6 mmol. l(-1) min(-1), P = not significant [NS] by ANOVA). Culture of SHR or WKY VSMCs in low [K(+)] media did not alter NHE-1 protein expression, suggesting the enhancement of activity in SHR cells was due to an increased turnover number of NHE-1. This response of NHE-1 in SHR VSMCs to K(+) depletion indicated a direct effect on these cells and could potentially enhance the contractile or proliferative phenotype of these cells in vivo.

Red cell Na+/Li+ countertransport and Na+/H+ exchanger isoforms in human proximal tubules.

BACKGROUND: Increased activity of the Na+/Li+ countertransporter (SLC) is a well-recognized intermediate phenotype of hypertension and diabetic nephropathy and may indicate a predisposition to hypertension. Previous work has attempted to link this membrane transport marker to altered Na+ reabsorption in the proximal tubule. Since the Na+/H+ exchanger (NHE) isoforms 1 and 3 are expressed in the basolateral and apical membranes of the proximal tubule, respectively, we investigated the relationship between these transport proteins and red cell SLC to examine whether the peripheral blood transport phenotype is associated with altered levels of transport proteins in the proximal tubule. METHODS: Proximal tubules were prepared from human nephrectomy specimens. NHE-1 and NHE-3 were detected on Western blots by specific antibodies. Red cell SLC was also measured. RESULTS: Both NHE-1 and NHE-3 proteins were demonstrated, with molecular weights of 97 and 85 kD, respectively. SLC was very strongly correlated with the level of NHE-3 protein (r = 0.78, P < 0.001) and was negatively related to NHE-1 protein (r = -0.32). In multiple regression analysis, only NHE-3 and NHE-1 protein levels were significant predictors of red cell SLC, accounting for up to about 70% of the variance of this parameter. CONCLUSIONS: We conclude that red cell SLC may be a marker of increased NHE-3 protein expression in the proximal tubule, which may account for the blunted pressure natriuresis and predisposition to hypertension.

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat.

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)/H(+) exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar-Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 degrees C, but growth is abolished at the restrictive temperature of 39 degrees C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H(+) efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0+/-2.8 and 39. 9+/-5.7 mmol.min(-1).l(-1) respectively; P<0.001; n=6). H(+) efflux due to non-Na(+)-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of (32)P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes.

The presence of endogenous phospholipase A (PL-A) activity of U. urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas. Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated. The pH optimum was about 8.5-9 for phospholipase A1 (PL-A1) in the three serovars. A more acidic pH optimum of 6 was observed for phospholipase A2 (PL-A2) enzymes in serovars 3 and 4. However, a very significant stimulation of PL-A2 activity in serovar 8 occurred around pH 7. The specific activity of PL-A2 was always 50-100 fold higher than PL-A1 activity in the pH range studied. Ca2+ ions only slightly stimulated PL-A1 activity in all 3 serovars. PL-A1 activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8. Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A2 activity in serovar 4. EDTA inhibition corresponded to Ca2+ stimulation for PL-A1 activity for serovars 3 and 8. A general stimulation of PL-A1 activity by diethyl ether was evident but the degree of stimulation varied with the serovar. Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8. PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme. All 3 serovars were inactivated by heat. A broad pH optimum for PL-C activity was evident around 7-8. Diethyl ether enhanced PL-C activity of serovar 8. Sodium deoxycholate and heat were inhibitory to PL-C activity. The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria. However, the major differences in the specific characteristics of specially PL-A1 and PL-A2 suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria. Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival. The ureaplasma PL-As are also markedly different from one serovar to another. This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus. There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta. There are marked differences in calcium concentrations under specific circumstances in various host tissues. Thus the differences in specific activity among the phospholipases of the serovars of U. urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.

Uptake mechanisms for ascorbate and dehydroascorbate in lymphoblasts from diabetic nephropathy and hypertensive patients.

In diabetic nephropathy and hypertension, a major cause of mortality is from cardiovascular disease. Since low levels of antioxidants such as vitamin C have been associated with such complications, we have examined the uptake mechanisms for ascorbic acid (AA) and dehydroascorbic acid (DHA) in lymphoblasts from normal control subjects (CON), normoalbuminuric insulin-dependent diabetic (IDDM) patients (DCON), patients with IDDM and nephropathy (DN) and hypertensive patients (HT) using mass assays of uptake and measuring AA using high-performance liquid chromatography. Precautions were taken to prevent oxidation of AA and to take into account the instability of DHA in buffers. DHA uptake was the major mechanism in all four groups of subjects, and the Vmax (maximal uptake rate) was significantly lower in the DN cells (24.7 +/- 1.0 nmol [95% confidence intervals CI 22.5, 26.3] 10(6) cells(-1) h(-1)) compared to CON and DCON cells (33.9 +/- 2.1 [95% CI 29.4, 38.4] and 37.0 +/- 2.2 [95% CI 32.2, 41.8] nmol 10(6) cells(-1) h(-1), respectively, p < 0.001 for both). DHA Vmax was also lower in the HT group (23.2 +/- 1.1 [95% CI 20.7, 25.7] nmol 10(6) cells(-1) h(-1)) compared to the CON group (p < 0.001). There were no significant differences in the Km or passive membrane permeability for DHA or the AA uptake. DHA uptake showed a negative correlation to systolic blood pressure (r(s) = -0.49, p < 0.001). These findings suggest that impaired DHA uptake may be one component of the phenotype expressed by DN cells that may persist in culture. Impaired DHA uptake in vivo, especially in the presence of hyperglycaemia, leads to impaired regeneration of AA and depletion of anti-oxidant defences, exposing such individuals to increased risk of cardiovascular disease.

Activity and expression of Na(+)-H+ exchanger isoforms 1 and 3 in kidney proximal tubules of hypertensive rats.

Increased activity of the cellular Na(+)-H+ exchangers (NHEs), especially isoform 1 (NHE-1), is a recognized intermediate phenotype of hypertension. NHE activity has been demonstrated to be increased in proximal tubules of the spontaneously hypertensive rat (SHR). However, with the recent cloning of other members of this family of transporters, it is unclear which isoforms may contribute to this increased activity. We have used specific antibodies raised against glutathione-S-transferase fusion proteins of rat NHE-1 and NHE-3 to determine the relative contributions of these isoforms to the NHE activity in freshly isolated and cultured proximal tubule cells from SHR and Wistar-Kyoto (WKY) normotensive control rats. In freshly isolated proximal tubule cells, NHE activity was elevated almost 3-fold in SHR cells (P < .001), and in both rat strains, the contribution from NHE-1 and NHE-3 was approximately equal. Western blots of membranes from these cells showed equal amounts of NHE-1 protein in SHR and WKY cells. However, NHE-3 protein expression was increased 50% in SHR cells (P < .001), and this may account for the elevated activity of this isoform in SHR. The effect of culturing these cells in vitro was then examined. Although total NHE activity in both cell types was decreased during culture, this was mainly due to loss of expression of NHE-3 protein. NHE-1 activity was persistently elevated in the SHR cells in culture. These findings suggest that elevated NHE activity in SHR proximal tubules could be mediated by two mechanisms: (1) increased NHE-1 activity without any increased NHE-1 protein content that persists despite culture and may resemble those changes described for extrarenal tissues and (2) increased NHE-3 activity due to increased expression of NHE-3 protein. Disappearance of NHE-3 during culture implies that our culture conditions did not replicate the in vivo environment and may have removed the factors contributing to the increased NHE-3 expression in SHR cells.

Calcium-induced activation of the rat vascular myocyte Na+/H+ exchanger isoform-1.

An established intermediate phenotype of human hypertension and diabetic nephropathy is an elevation of Na+/H+ exchanger (NHE) activity, but the mechanism for this is unclear. This phenotype is maintained in vascular myocytes from the spontaneously hypertensive rat (SHR) compared with the normotensive Wistar Kyoto rat (WKY). Since intracellular calcium levels ([Ca2+]i) following agonist stimulation were elevated in cells from both hypertensive humans and SHR, we have examined the role of calcium-calmodulin (CaM) in the mechanism of increased NHE activity in vascular myocytes of SHR by determining the activity and phosphorylation state of NHE isoform-1 (NHE-1) in cells from SHR and WKY when [Ca2+]i was elevated by the ionophores A23187 or ionomycin. NHE activity was measured using fluorometry and NHE-1 phosphorylation by immunoprecipitating the exchanger from 32P-orthophosphate-labeled cells with a polyclonal NHE-1-specific antibody. The ionophore A23187 increased [Ca2+]i in both cell types to approximately 700 to 800 nmol x L(-1), and led to stimulation of NHE-1 activity only in WKY myocytes, with no effect on SHR cells. An inhibitor of CaM kinase II (KN-62) failed to abolish stimulation of NHE-1 by A23187 in WKY cells, and had no effect on unstimulated NHE-1 activity in both cell types. Ionomycin also elevated [Ca2+]i in both cell types to approximately 1,000 nmol x L(-1) and activated NHE-1 activity in only WKY cells. Activation of NHE-1 in WKY cells by an increased [Ca2+]i was not mediated by an increase in NHE-1 phosphorylation, whether in the presence or absence of KN-62. The elevated NHE-1 phosphorylation in SHR cells was not affected by elevated [Ca2+]i or KN-62. Calmodulin-agarose beads bound NHE-1 extracted from SHR cells to a lesser extent than that from WKY cells. We conclude that calcium-induced NHE-1 activation in WKY cells was not mediated by CaM kinase II. The elevated NHE-1 activity and phosphorylation of SHR cells was not further modulated by increased [Ca2+]i, and was also independent of CaM kinase II. Non-phosphorylation-dependent mechanisms of activation of NHE-1 may therefore be responsible for alterations of NHE-1 activity in these cells, such as the direct binding of CaM to NHE-1. This direct binding of CaM to NHE-1 may be impaired in SHR compared with WKY cells.

Enhanced mitogen-activated protein kinase activity and phosphorylation of the Na+/H+ exchanger isoform-1 of human lymphoblasts in hypertension.

Increased activity of the Na+/H+ exchanger isoform-1 (NHE-1) is recognized as an intermediate phenotype for hypertension, but the basis for this association is unclear. We have previously demonstrated an increased phosphorylation of NHE-1 in lymphoblasts from hypertensives that was associated with increased cell proliferation. Due to the central importance of mitogen-activated protein kinases (MAPKs) in signaling cascades transducing responses from extracellular growth factors and hormones, we examined the activity of this kinase in a specific peptide phosphorylation assay. Cells from hypertensives showed a significant twofold enhancement of MAPK activity (P < .001). This was not associated with any increase in p42mapk and p44mapk protein content. There was no significant increase in the level of tyrosine phosphorylation of MAPK in cells from hypertensives. MAPK activity was correlated with NHE-1 activity (r(s) = .55, P < .01) and phosphorylation (r(s) = .51, P < .02). These findings suggest that the increased cell proliferation rate, NHE-1 activity, and phosphorylation of lymphoblasts from hypertensives may be associated with enhanced MAPK activity, suggesting upregulation of this signaling pathway in hypertension.

Phosphorylation and activity of Na+/H+ exchanger isoform 1 of immortalized lymphoblasts in diabetic nephropathy.

In both essential hypertension and diabetic nephropathy (DN), the ubiquitous cellular Na+/H+ exchanger (NHE) exhibits altered kinetics with increased transport activity. The mechanism for this phenotype and its dependence on the presence of serum are unknown, but increased lymphoblast NHE activity in DN has been attributed to a defect in post-translational processing of NHE-1 rather than an increased cellular exchanger number. Phosphorylation of NHE-1 has been proposed to play a role in its activation in a variety of cell models. We have examined, therefore, the role of NHE-1 phosphorylation and the effect of serum in determining the increased NHE-1 activity in lymphoblasts from patients with DN. Cells from these patients exhibited increased NHE activity in the presence and absence of fetal calf serum (range 42-59%, P < 0.005, analysis of variance) and an increased proliferation rate (P < 0.01) when compared with cells from both normoalbuminuric diabetic patients and non-diabetic control subjects. However, NHE-1 abundance was very similar among all groups in the presence and absence of serum, indicating that increased NHE activity in cells of nephropathy patients was due to an increased turnover number. This nephropathy phenotype was not accompanied by an increased net phosphorylation of NHE-1 in the presence or absence of serum. Our findings suggest that increased NHE-1 activity in cells of DN patients is independent of the presence of serum and is not attributable to altered NHE-1 phosphorylation. Additional post-translational mechanisms for activation of NHE-1, therefore, may be involved.

Na(+)-H+ antiporter phenotype, abundance, and phosphorylation of immortalized lymphoblasts from humans with hypertension.

Previous studies have demonstrated an elevated Na(+)-H+ exchanger activity in various cell types from patients with essential hypertension. The phenotype of an increased maximal transport capacity is preserved in Epstein-Barr virus immortalized lymphoblasts from hypertensive patients. The mechanisms underlying this abnormality are unclear. In this study, we used lymphoblasts from hypertensive patients and normotensive control subjects with and without a family history of hypertension to determine (1) Na(+)-H+ exchanger activity using fluorometry with the pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, (2) Na(+)-H+ exchanger isoform 1 abundance with specific polyclonal antibodies, and (3) Na(+)-H+ exchanger phosphorylation by immunoprecipitation of the 32P-labeled transporter. Na(+)-H+ exchanger activity (in millimoles per liter per minute) measured when pHi was clamped at 6.0 was significantly higher in cells from hypertensive patients (18.8 +/- 0.6, P < .001) and those subjects with a family history of hypertension (16.4 +/- 0.6, P < .001) compared with normotensive control subjects (12.9 +/- 0.6). Exchanger abundance was identical in all three groups of subjects, indicating that increased activity in the hypertensive group was due to an elevated turnover number of the exchanger. Na(+)-H+ exchanger phosphorylation in quiescent cells was significantly elevated in cells from hypertensive patients (1.58 +/- 0.16, P < .001) compared with control subjects (1.00 +/- 0.07), and cells from normotensive subjects with a hypertensive family history showed intermediate values (1.23 +/- 0.14). Identical changes in Na(+)-H+ exchanger function and phosphorylation have been demonstrated in vascular smooth muscle cells from spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose-induced changes in turnover of Na+/H+ exchanger of immortalized lymphoblasts from type I diabetic patients with nephropathy.

Increased cellular Na+/H+ exchanger (NHE) activity has been demonstrated in type I diabetic patients with nephropathy. Such patients also have a previous history of poor glycemic control. The interaction between hyperglycemia and changes in NHE activity remains obscure. Therefore, we examined the effects of media containing 5 and 25 mmol/l glucose on the increased NHE activity and turnover number in Epstein-Barr virus-transformed lymphoblasts from patients with diabetic nephropathy compared with normoalbuminuric diabetic and nondiabetic control subjects. NHE activity was determined fluorometrically, and NHE isoform 1 (NHE-1) density was measured with specific polyclonal antibodies. In the presence of 5 mmol/l glucose, cells from patients with diabetic nephropathy exhibited higher NHE activity with intracellular pH clamped to 6.0 compared with diabetic and nondiabetic control subjects (P < 0.005 for both), due to a higher turnover number of NHE-1. Incubation in 25 mmol/l glucose for 48 h caused an increase in NHE activity (P < 0.001) and turnover number (P < 0.01) in the diabetic nephropathy group only, with no significant change in the diabetic or nondiabetic control groups. The rate constants for cell proliferation and NHE activity or turnover number were correlated when cells were cultured in 5 mmol/l glucose (r = 0.34 and 0.32, respectively; P < 0.05) or 25 mmol/l glucose media (r = 0.66 and 0.65, respectively; P < 0.001). We conclude that only lymphoblasts from the diabetic nephropathy group show an increase in NHE activity and turnover number under conditions mimicking hyperglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Mycoplasma arthritidis superantigen on enzymatically induced arthritis in mice.

The objective of this study was to examine the effect of the stimulation of the immune system with Mycoplasma arthritidis superantigen (MAS) on joint inflammation and cartilage destruction. MAS was administered either alone or combined with a model of degenerative arthritis induced by intraarticular injection of collagenase enzyme. Intraperitoneal injection of MAS resulted in activation of peripheral lymphocytes in BALB/c mice, as shown by a proliferative response of splenocytes isolated from MAS-treated animals to IL-2-containing supernatant. Intraperitoneal or intra-articular administration of MAS alone at concentrations maximally activating lymphocytes had no detectable effect on joints. Intra-articular injection of collagenase resulted in some infiltration of inflammatory cells into the joints, hyperplasia and hypertrophy of synovial lining, pannus formation and surface loss of proteoglycans 7 days following the injection. At 21 days, the animals showed almost total loss of cartilage and minimal or no inflammation. Animals receiving MAS in addition to collagenase treatment showed similar changes in the joints. These data have demonstrated that activation of the immune system with MAS in vivo does not increase joint inflammation or cartilage degradation in enzymatically induced arthritis.

Abnormal Na+/H+ antiporter phenotype and turnover of immortalized lymphoblasts from type 1 diabetic patients with nephropathy.

Cellular Na+/H+ exchanger (NHE) activity is elevated in type 1 diabetic patients with nephropathy and patients with essential hypertension. The characteristics of this NHE phenotype in hypertension (raised Vmax and a lowered Hill coefficient) are preserved in Epstein-Barr virus-transformed lymphoblasts from hypertensive patients. In this study, we have determined NHE kinetics in cultured lymphoblasts from diabetic patients with and without nephropathy, with nondiabetic controls, using fluorometry with the pH indicator 2,7'-bis-(carboxyethyl)-5,6-carboxyfluorescein and estimation of NHE isoform 1 (NHE-1) density with specific polyclonal antibodies. The Vmax of NHE was elevated significantly, and the Hill coefficient for internal H+ binding was lowered in cells from patients with diabetic nephropathy compared with both normal controls and normoalbuminuric diabetic patients. NHE-1 density as measured by Western blotting was similar in all groups. The turnover number of NHE-1 was thus elevated in cells from nephropathy patients. This phenotype in cells from diabetic nephropathy patients resembles that in essential hypertension and suggests that such patients may have a predisposition to hypertension. Moreover, as these changes persist in cultured lymphoblasts in vitro, these cells should provide a cell culture model to further define the basic mechanisms leading to NHE activation in diabetic nephropathy.

Ureaplasma diversum infection in vitro alters prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells without affecting cell viability.

Bovine epithelial and stromal cells of the endometrium were inoculated with Ureaplasma diversum, pathogenic strain 2312, at 10(6) or 10(3) color-changing units (ccu)/ml in the presence of 1% fetal bovine serum (depleted of steroids by dextran-charcoal treatment) to assess the effect of infection on prostaglandin biosynthesis. When the inoculum of U. diversum was 10(6) ccu/ml, the concentration of U. diversum in the culture medium decreased with time. U. diversum was found on the epithelial and stromal cell monolayers, increasing in titer 100-fold, indicating that attachment and eventually growth occurred. When the inoculum was 10(3) ccu/ml, the titer of U. diversum remained the same or increased in the supernatant and increased on epithelial and stromal cells. The effect of infection was evaluated by measurement of the primary prostaglandin produced by each cell type, prostaglandin F2a for epithelial cells and prostaglandin E2 for stromal cells. Infection with U. diversum significantly decreased prostaglandin F2a accumulation, by 44.7% +/- 6.0% at 10(6) ccu/ml (P < or = 0.005) and 15.8% +/- 5.3% at 10(3) ccu/ml (P < or = 0.05) in epithelial cells. Prostaglandin E2 accumulation by stromal cells was decreased by 34.0% +/- 4.0% at 10(6) ccu/ml (P < or = 0.001) and by 13.5% +/- 2.7% at 10(3) ccu/ml (P < or = 0.005). Infection with 10(6) ccu/ml did not alter endometrial cell viability, as shown by protein measurement, trypan blue dye exclusion, and cell plating efficiency tests. Thus, alterations in prostaglandin production were not due to cell deterioration. These observations suggest that U. diversum can alter prostaglandin E2 and prostaglandin F2a patterns in primary cultures of bovine endometrial cells without affecting cell viability.

Serological response to Ureaplasma urealyticum in the neonate.

A serovar-specific antibody response to Ureaplasma urealyticum was observed in stillborns, neonates, and mothers by means of the modified metabolic inhibition test. Elevated levels of ureaplasma antibody were found in cases of stillbirth and neonatal respiratory disease. There was a higher risk of mortality among neonates with respiratory disease who had elevated levels of ureaplasma antibody. IgG and IgM serovar-specific antibody responses to U. urealyticum serovars were confirmed by the immunoperoxidase and immunofluorescence assays. In contrast to ELISA-based assays that use soluble membrane proteins as antigen, these three assays use whole intact cells. These observations suggest that the serovar-specific antibody response may be associated with the ureaplasma extramembranous carbohydrate (lypoglycan) coat, which comes off during centrifugation. With the immunoperoxidase assay, a heel-prick blood sample (20 microL) can be screened for 16 IgM or IgG + IgM + IgA (combined) responses in 4 hours. The predictive value of IgM antibody for U. urealyticum infection and other mycoplasma infections and for the risk of stillbirth and development of chronic lung disease should now be assessed.

Localization of endogenous activity of phospholipases A and C in Ureaplasma urealyticum.

Endogenous activities of phospholipases A and C in Ureaplasma urealyticum were assayed in cellular fractions of exponential-phase cells. Enzymatic studies indicated that ATPase activity was localized in the plasma membrane fraction and NADH and NADPH dehydrogenase activities were localized in the cytosol fraction. Studies with purified ureaplasma membranes demonstrated that, of three serovars tested, endogenous phospholipase A1, A2, and C activities were localized in the plasma membrane. Very low levels of activity were observed in the cytosol fractions. Phospholipase A2 activity in the plasma membrane was 3- to 5-fold higher than the activity in the lysates and 60- to 300-fold higher than the activity of phospholipase A1. Phospholipase C was localized mainly in the plasma membrane, with 20% found in the cytosol fraction. The levels of activity were comparable among the three serovars. There was a significantly lower level of activity in cells from the stationary growth phase than in the exponential phase. Significant differences were observed in the phospholipase A activities among the U. urealyticum serovars 3, 4, and 8. Phospholipase A2 activity was twofold higher in serovar 8 membranes, and phospholipase A1 activity was twofold higher in serovar 3 membranes. These results demonstrate that endogenous activities of phospholipase A and C are localized primarily in the plasma membrane fraction of U. urealyticum. The specific activities in the membranes of the phospholipases varied among the three serovars. Phospholipase enzymes may function as virulence factors in U. urealyticum and may vary among the serovars.

Common sulfoglycolipid receptor for mycoplasmas involved in animal and human infertility.

Sulfoglycolipids are ubiquitous components of the male germ cell membrane. Sulfogalactoglycerolipid (SGG) is restricted to mammalian cells and has recently been implicated in sperm/egg interactions. Mycoplasma infections have been implicated in infertility in a variety of species, including humans. Four such species-specific mycoplasmas, Ureaplasma urealyticum and Mycoplasma hominis (humans), Mycoplasma pulmonis (rodents), and Ureaplasma diversum (cattle) are not shown to specifically recognize SGG and the sphingolipid counterpart, sulfogalactosyl ceramide. This glycolipid receptor binding may relate to the reproductive pathogenesis of these organisms.