Correlation of cystatin-C with glomerular filtration rate by inulin clearance in pregnancy.

OBJECTIVE: To test utility of cystatin-C as a marker of glomerular filtration rate during pregnancy, we performed serial correlations with inulin clearance during pregnancy and postpartum. METHODS: Twelve subjects received inulin infusions and serum cystatin-C at three time points. Pearson's correlation coefficient was calculated. RESULTS: Cystatin-C levels ranged 0.66-1.48 mg/L during pregnancy, and 0.72-1.26 mg/L postpartum. Inulin clearance ranged 130-188 mL/min during pregnancy, and 110-167 mL/min postpartum. Cystatin-C did not correlate with inulin clearance at any time point. CONCLUSION: Serum cystatin-C did not correlate with inulin clearance during pregnancy or postpartum.

PP087. Deep trial secondary analysis: Digoxin immune fab fragment treatment has additional benefits in endogenous digitalis-like factor positive preeclamptic women.

INTRODUCTION: A double blinded placebo controlled clinical trial of a commercial digoxin immune Fab fragment (DIF) in preeclamptic (PE) women provided some benefit to treated subjects (1). In that study DIF, relative to placebo, prevented a decline in CrCl and lowered levels of endogenous digitalis-like factor (EDLF) activity as measured by sodium pump inhibition (SPI). However, some PE subjects had undetectable EDLF. OBJECTIVES: The hypothesis tested was that only PE women with measureable EDLF would respond to DIF treatment and analysis of EDLF positive women might reveal treatment effects masked by inclusion of EDLF negative, and hence non-responding, PE women. Accordingly, analyses of DIF effects in EDLF positive PE women were conducted. METHODS: Patient characteristics and study design have been published (1). In these subanalyses, subjects were considered to be EDLF positive if their plasma inhibited red cell sodium pump mediated Rb uptake. All analyses were redone for the EDLF positive subgroup by Covance Inc as in the original trial. Continuous data were analyzed by ANCOVA. Categorical data were analyzed by Barnard Exact Test. RESULTS: 45 subjects (23 DIF, 22 placebo) had baseline SPI evaluated. Of these 22% had undetectable SPI. EDLF positive PE women showed greater and more significant reductions of SPI in response to DIF at each time point (12, 24, 48 hr treatment) than in the original analysis. Subjects with undetectable EDLF showed no significant change in response to DIF or placebo. For CrCl, EDLF positive PE women showed greater and more significant preservation of CrCl compared with original analyses. Subjects absent EDLF showed deterioration of CrCl with or without DIF. Among EDLF positive PE women DIF treated women had significantly less maternal pulmonary edema (p=0.035) and significantly less intraventricular hemorrhage in their infants (p=0.015). There was the suggestion of reductions in the incidence of other maternal and neonatal abnormalities. CONCLUSION: These data indicate that EDLF positive PE women are those that responded to DIF and also raise the possibility of extended benefits of DIF treatment in this group. Results support further research in this area.

Toxicokinetics of bis(2-chloroethoxy)methane following intravenous administration and dermal application in male and female F344/N rats and B6C3F1 mice.

In the National Toxicology Program's toxicity studies, rats were more sensitive than mice to Bis(2-chloroethoxy)methane (CEM) - induced cardiac toxicity following dermal application to male and female F344/N rats and B6C3F1 mice. Thiodiglycolic acid (TDGA) is a major metabolite of CEM in rats. It has been implicated that chemicals metabolized to TDGA cause cardiac toxicity in humans. Therefore, the toxicokinetics of CEM and TDGA were investigated in male and female F344/N rats and B6C3F1 mice following a single intravenous administration or dermal application of CEM to aid in the interpretation of the toxicity data. Absorption of CEM following dermal application was rapid in both species and genders. Bioavailability following dermal application was low but was higher in rats than in mice with females of both species showing higher bioavailability than males. CEM was rapidly distributed to the heart, thymus, and liver following both routes of administration. Plasma CEM C(max) and AUC(∞) increased proportionally with dose, although at the dermal dose of 400mg/kg in rats and 600mg/kg in mice non-linear kinetics were apparent. Following dermal application, dose-normalized plasma CEM C(max) and AUC(∞) was significantly higher in rats than in mice (p-value<0.0001 for all comparisons except for C(max) in the highest dose groups where p-value=0.053). In rats, dose-normalized plasma CEM C(max) and AUC(∞) was higher in females than in males: however, the difference was significant only at the lowest dose (p-value=0.009 for C(max) and 0.056 for AUC(∞)). Similar to rats, female mice also showed higher C(max) and AUC(∞) in females than in male: the difference was significant only for C(max) at the lowest dose (p-value=0.002). Dose-normalized heart CEM C(max) was higher in rats than in mice and in females than their male counterparts. The liver CEM C(max) was lower compared to that of heart and thymus in both rats and mice following intravenous administration and in rats following dermal application. This is likely due to the rapid metabolism of CEM in the liver as evidenced by the high concentration of TDGA measured in the liver. Dose-normalized plasma and heart TDGA C(max) values were higher in rats compared to mice. In rats, females had higher plasma and heart TDGA C(max) than males; however, there was no gender difference in plasma or heart TDGA C(max) in mice. These findings support the increased sensitivity of rats compared to mice to CEM-induced cardiac toxicity. Data also suggest that, either CEM C(max) or AUC can be used to predict the CEM-induced cardiac toxicity. Although, both plasma and heart TDGA C(max) was consistent with the observed species difference and the gender difference in rats, the gender difference in mice to cardiac toxicity could not be explained based on the TDGA data. This animal study suggests that toxicologically significant concentrations of CEM and TDGA could possibly be achieved in the systemic circulation and/or target tissues in humans as a result of dermal exposure to CEM.

Toxicology and carcinogenesis studies of microencapsulated trans-cinnamaldehyde in rats and mice.

trans-Cinnamaldehyde is a widely used natural ingredient that is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F(1) mice were exposed to microencapsulated trans-cinnamaldehyde in the feed for three months or two years. All studies included untreated and vehicle control groups. In the three-month studies, rats and mice were given diets containing 4100, 8200, 16,500, or 33,000 ppm trans-cinnamaldehyde. In rats, feed consumption was reduced in all exposed groups. In mice, feed consumption was reduced in the highest dose groups. Body weights of all treated males were less than controls. Body weights were reduced in female rats exposed to 16,500 or 33,000 ppm and female mice exposed to 8200 ppm or greater. All rats survived to the end of the study but some male mice in the highest dose groups died due to inanition from unpalatability of the dosed feed. The incidence of squamous epithelial hyperplasia of the forestomach was significantly increased in rats exposed to 8200 ppm or greater and female mice exposed to 33,000 ppm. In mice, the incidence of olfactory epithelial degeneration of the nasal cavity was significantly increased in males and females exposed to 16,500 ppm and females exposed to 33,000 ppm. In the two-year studies, rats and mice were exposed to 1000, 2100, or 4100 ppm trans-cinnamaldehyde. Body weights were reduced in mice exposed to 2100 ppm and in rats and mice exposed to 4100 ppm. In rats, hippuric acid excretion was dose proportional indicating that absorption, metabolism, and excretion were not saturated. No neoplasms were attributed to trans-cinnamaldehyde in rats or mice. Squamous cell papillomas and carcinomas of the forestomach were observed in male and female mice but the incidences were within the NTP historical control range and were not considered to be related to trans-cinnamaldehyde exposure.

Digitalis-like factor response to hyperinsulinemia accompanying a euglycemic hyperinsulinemic clamp or oral glucose tolerance test.

Many studies of essential hypertension find evidence of insulin resistance in the same individuals, leading some to postulate a hypertensive role for insulin. However, the mechanisms by which insulin might exert a hypertensive effect are not fully resolved. An endogenous sodium pump inhibitor or digitalis-like factor (DLF) has been proposed as a hypertensive agent and its plasma concentrations are elevated in hypertension and in Type II diabetes, where insulin levels are elevated. Hence, we studied the effect of insulin on DLF using two approaches to achieve hyperinsulinemia. Normotensive men and women underwent a hyperinsulinemic, euglycemic clamp (40 mU/m2/min insulin, 40 mU = 1.6 x 10(-6) g) in which plasma insulin concentration was kept at high, but physiologic levels. Serum DLF (measured as inhibition of [Na,K]ATPase activity) and insulin levels were measured at baseline and every 30 min throughout the 2 hr clamp. Additionally, other subjects underwent an oral glucose tolerance test (OGTT) as a second means of increasing insulin levels. Insulin and DLF levels were measured prior to and hourly for 3 hours after receiving 100 gm of oral glucose. Serum DLF increased significantly during the clamp from a baseline of 4.6 +/- 0.81 to a peak of 8.7 +/- 1.2% inhibition (p=0.001). Comparison of the baseline and peak DLF levels with concomitant plasma insulin levels revealed a significant correlation (R=0.60, p=0.003). During the OGTT, DLF levels rose from a baseline of 2.4 +/- 1.0 to a peak level of 5.0 +/- 0.4%, p = 0.04. These results suggest that DLF, a factor that can cause vascular smooth muscle contraction and potentially influence blood pressure, is increased by hyperinsulinemia and provides a mechanism by which insulin may increase blood pressure.

A dynamic inline sample thermoregulation unit for flow cytometry.

BACKGROUND: Flow cytometry is a valuable tool for the study of cellular and molecular interactions. We sought to develop instrumentation that would allow accurate and precise inline dynamic temperature control of flow cytometry samples in order to expand the analytical capabilities of flow cytometry. METHODS: Using a temperature controller, DC power supply, and a Peltier module, we designed a temperature control circuit that regulates the temperature of a heat transfer block. The heat transfer block surrounds the sample line to efficiently heat and cool the sample. We attached DNA oligomers to microspheres and observed the denaturation of fluorescently labeled complementary oligomers to verify that the sample was achieving the desired temperature. RESULTS: The inline thermoregulation unit can ramp between 30 and 95 degrees C (>2 degrees C per second) within 30 s repeatedly. The accuracy of the instrument was verified by comparing the observed melting temperatures of the oligomer sets to the predicted values. These values varied within 6% of the predicted and were reproducible over a variety of conditions. CONCLUSIONS: The apparatus we constructed accurately and dynamically controls the sample temperature of inline samples. Flow cytometers equipped with our inline thermoregulation unit will be able to conveniently use temperature as a robust experimental parameter.

Two-year toxicity and carcinogenicity study of methyleugenol in F344/N rats and B6C3F(1) mice.

Methyleugenol (MEG) was tested for toxicity/carcinogenicity in a 2-yr carcinogenesis bioassay because of its widespread use in a variety of foods, beverages, and cosmetics as well as its structural resemblance to the known carcinogen safrole. F344/N rats and B6C3F(1) mice (50 animals/sex/dose group) were given MEG suspended in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg/day for 2 yr. Control groups (60 rats/sex and 50 mice/sex) received only the vehicle. A stop-exposure group of 60 rats/sex received 300 mg/kg/day by gavage for 53 weeks followed by the vehicle only for the remaining 52 weeks of the study. A special study group (10 animals/sex/species/dose group) were used for toxicokinetic studies. All male rats given 150 and 300 mg/kg/day died before the end of the study; survival of female rats given 150 mg/kg/day and all treated female mice was decreased. Mean body weights of treated male and female rats and mice were decreased when compared to control. Area under the curve results indicated that greater than dose proportional increases in plasma MEG occurred for male 150 and 300 mg/kg/day group rats (6 and 12 month) and male 150 mg/kg/day mice (12 month). Target organs included the liver, glandular stomach, forestomach (female rats) and kidney, mammary gland, and subcutaneous tissue (male rats). Liver neoplasms occurred in all dose groups of rats and mice and included hepatoadenoma, hepatocarcinoma, hepatocholangioma (rats only), hepatocholangiocarcinoma, and hepatoblastoma (mice only). Nonneoplastic liver lesions included eosinophilic and mixed cell foci (rats only), hypertrophy, oval cell hyperplasia, cystic degeneration (rats only), and bile duct hyperplasia. Mice also exhibited necrosis, hematopoietic cell proliferation, and hemosiderin pigmentation. Glandular stomach lesions in rats and mice included benign and malignant neuroendocrine tumors, neuroendocrine cell hyperplasia, and atrophy and in mice included glandular ectasia/chronic active inflammation. In female rats, the forestomach showed a positive trend in the incidences of squamous cell papilloma or carcinoma (combined). Male rats also exhibited kidney (renal tubule hyperplasia, nephropathy, and adenomacarcinoma), mammary gland (fibroadenoma), and subcutaneous tissue (fibroma, fibrosarcoma) lesions. Male rats also exhibited malignant mesotheliomas and splenic fibrosis. These data demonstrate that MEG is a multisite, multispecies carcinogen.

Sodium-dependent calcium release from vascular smooth muscle mitochondria.

Interest in mitochondrial calcium (Ca2+) uptake and release waned as it became apparent that sarcoplasmic reticulum calcium stores dominate the control of cytoplasmic calcium concentration. Our recent demonstration of a very large rise in vascular smooth muscle (VSM) cytoplasmic sodium (Na+) concentration after inhibition of the sodium, potassium-ATPase (sodium pump) led us to several questions. Do VSM mitochondria show Na(+)-dependent Ca2+ release? Are the documented changes in cytoplasmic Na+ concentration sufficient to cause Ca2+ release? Do features of the cardiac mitochondrial exchange system, including differential sensitivity to a number of calcium antagonists and cation specificity, apply to VSM? We isolated mitochondria from bovine aorta and mesenteric arteries and employed arsenazo III as the Ca2+ indicator. Mitochondria from arterial vessels accumulated added calcium (up to 50 nmol Ca2+/mg protein) and released Ca2+ on exposure to Na+. This concentration-dependent relationship was linear from 0 to 10 mM of Na+, and it plateaued between 20 mM and 40 mM of Na+. VSM mitochondria exposed to 20 mM Na+ released 118 +/- 25 nmol Ca2+ per mg mitochondrial protein in 20 min, when a new equilibrium was reached. Lithium (Li+), in contrast to Na+, produced much smaller amounts of Ca2+ release from the VSM mitochondria. Na+-dependent Ca2+ release was antagonized in a concentration-dependent manner by diltiazem (0-320 microM) with a Ki of 10.2 microM. Nifedipine had a lesser effect, and verapamil produced almost no inhibition. VSM mitochondria responses resemble those from heart mitochondria in that Na+-dependent Ca2+ release is present with a similar range of sensitivity to Na+ and a similar pattern of influence of diltiazem, nifedipine and verapamil. However, the influence of Li+ on Ca2+ release was much smaller and the amount of the Ca2+ released was much greater for VSM mitochondria compared with that reported for heart mitochondria. The large amount of Ca2+ released and the range of Na+ concentration that provoked Ca2+ release being within the physiologically achievable range raise the interesting possibility that these mechanisms may modify intramitochondrial cytosolic Ca2+ concentration, and hence could potentially contribute to the contractile response that follows inhibition of the sodium pump.

Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization.

We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase from cloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activity of the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit in this two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociation constant of 35 +/- 16 nM as measured by the concentration dependence of its effect in stimulating maximal DNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to every kinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication. The accessory protein makes the DNA binding 3.5-fold tighter (K(d) of 9.9 +/- 2.1 nM compared to 39 +/- 10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 +/- 0.001 compared to 0.03 +/- 0.001 s(-)(1)). The ground-state nucleotide binding is improved from 4.7 +/- 2.0 to 0.78 +/- 0.065 microM, and the maximum DNA polymerization rate is increased from 8.7 +/- 1.1 to 45 +/- 1 s(-)(1) by the addition of the accessory protein. This leads to an increase in processivity from an estimated 290 +/- 46 to 2250 +/- 162. Although the accessory protein has been described as a "processivity factor" because of its effect on the ratio of rate constants defining processivity, this terminology falls short of adequately describing the profound effects of the small subunit on nucleotide-binding and incorporation catalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with a comprehensive analysis of the structural and mechanistic determinants of enzyme specificity that govern toxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.

Application of supercritical fluid chromatography to characterize a labile digitalis-like factor.

A sodium pump inhibitor (digitalis-like factor), isolated from the peritoneal dialysate of volume-expanded, hypertensive patients with kidney failure who were treated with this dialysis modality, was further purified and characterized by means of supercritical fluid chromatography, a separation technique whose application to very-low-concentration biomolecules is new. Previous studies suggested that after high-performance liquid chromatography (HPLC) purification, this inhibitor was the only factor correlated with volume status and blood pressure in these patients. When this same HPLC fraction was furthered purified on 2-dimensional supercritical fluid chromatography, a single peak coeluted with [Na,K]ATPase inhibitory activity. When split specimens were used, there was a strict correlation between the peak area, measured by flame ionization detection, and activity (n=10, R=0.98, P=0.00001). Inhibitory activity after supercritical fluid chromatography was still correlated with the degree of volume expansion of donor patients (P=0.01). After HPLC purification, this volume-sensitive inhibitor was chemically labile. With further purification on supercritical fluid chromatography, the active peak was still labile with comparable half-life. Supercritical fluid chromatography coupled with flame ionization detection provided an estimate of the amount of the inhibitor present. Again using split specimens, we determined that the labile digitalis-like factor was approximately 30-fold more effective than ouabain in inhibiting renal [Na,K]ATPase activity and >/=500 times more effective than ouabain in causing vascular smooth muscle contraction. The data suggest that we have purified to homogeneity a labile digitalis-like factor that is readily distinguished from ouabain or bufalin, based on chromatographic characteristics, chemical lability, and a much lower effective concentration for its biological activity.

Sodium pump inhibition and regional expression of sodium pump alpha-isoforms in lens.

Both hypertension and cataract formation have been associated with reductions in sodium pump activity, possibly as a result of an endogenous inhibitor. The objective of the present study was to answer 4 closely related questions: (1) Is the lens sodium pump effectively inhibited by a labile, digitalis-like factor we have identified in the peritoneal dialysate from hypertensive patients in end-stage renal failure? (2) How does that inhibition compare to that induced by ouabain? (3) Does sodium pump isoform distribution determine the degree of lens sodium pump inhibition? (This question was precipitated by the unanticipated finding that the labile DLF was more effective in inhibiting lens sodium pump than was anticipated.) (4) Is sodium pump activity altered in lens in response to increased salt intake, a maneuver known to increase endogenous digitalis-like factor? We found that whereas ouabain produced equivalent or significantly less inhibition of lens Na(+), K(+)-ATPase from calf or rabbit, respectively, compared with brain, labile digitalis-like factor preferentially inhibited lens compared with brain. Analysis of whole-lens preparations from rabbit, calf, and normal human lens revealed substantial alpha2- and alpha3-isoforms of the sodium pump but little alpha1-isoform. Ouabain inhibition of whole-lens Na(+),K(+)-ATPase from rabbit and calf were comparable: for rabbit lens, K(i)=5.2x10(-7) mol/L; for calf lens, K(i)=1.0x10(-6) mol/L. Limited quantities of labile digitalis-like factor prohibited similar determinations; however, its concentration-activity profile paralleled that of ouabain. Na(+), K(+)-ATPase activity, measured in the 3 major anatomic regions of lens and normalized to nucleus, was greatest in epithelium (56. 9+/-17.9) compared with cortex (5.8+/-1.4) and nucleus (1.0+/-0.0; P=0.01). Immunohistochemistry of rabbit lens found abundant alpha2- and alpha3-isoforms in epithelium and limited alpha3 but undetectable alpha1 in cortex and nucleus. Finally, rats randomized to a high Na diet showed significantly reduced lens Na(+), K(+)-ATPase activity compared with those on a low Na diet, consistent with the effects of a sodium pump inhibitor. In conclusion, the present study suggests that digitalis-like factor may provide a link between hypertension and cataract formation.

Higher cholesterol and insulin levels in pregnancy are associated with increased risk for pregnancy-induced hypertension.

Hyperinsulinemia and dyslipidemia are known to be associated with essential hypertension but their role in pregnancy-induced hypertension remains unclear. We performed a case-control study comparing cholesterol, insulin, and glucose levels in the early third trimester of pregnancy among 31 women who developed pregnancy-induced hypertension (PIH) (either preeclampsia [n = 6] or nonproteinuric gestational hypertension [n = 25]), with 31 women remaining normotensive through pregnancy. As compared with women remaining normotensive, women subsequently developing PIH had higher fasting cholesterol levels (279 v 247 mg/dL; P = .02) and higher fasting insulin levels (13.3 v 7.9 microU/mL; P = .03), although fasting glucose levels and levels of glucose and insulin after glucose load did not differ significantly between groups. In comparing hypertensive subgroups, fasting insulin levels were significantly higher among women who subsequently developed preeclampsia, but not among those subsequently developing nonproteinuric gestational hypertension. Although women developing PIH had higher pregravid body mass index (25.1 v 22.6 kg/m2, P = .06), fasting cholesterol and insulin levels were associated with risk for PIH even after adjustment for body mass index and age (relative risks for one unit increase, respectively: 1.02 (P = .03) and 1.12 (P = .03). Higher fasting cholesterol and insulin levels in mid- to late pregnancy are associated with increased risk for PIH. These observations support a role for insulin resistance in the development of this complication of pregnancy.

Eclampsia: a 13-year experience at a United States tertiary care center.

Eclampsia, the occurrence of a grand mal seizure in the setting of hypertension in pregnancy, remains a major women's health issue and an important cause of maternal and fetal morbidity in the United States. We reviewed the incidence, management, and outcome of cases of eclampsia during a 13-year period at a major maternity hospital. We confirmed 33 cases of eclampsia seen during that period and have evaluated risk factors in this population. Medical records were reviewed to obtain demographic and clinical data. Characteristics of the eclamptic women were compared with those of the general obstetric population during the same time period. The overall incidence of eclampsia at this tertiary care center was 0.028%. The majority of eclamptic women (75%) had four or more prenatal visits. Young age (< or = 20 years) and first pregnancy remained important risk factors for eclampsia. Although many women with eclampsia had preceding hypertension or elevated urine protein levels or both, some experienced eclampsia as their first disease manifestation. Although the occurrence of eclampsia was low, eclampsia continues to complicate pregnancy in this large U.S. obstetric population.

Regulation of the sodium pump in pregnancy-related tissues in preeclampsia.

OBJECTIVE: This study examined the expression of the three alpha-isoforms of the sodium pump in preeclampsia. Reductions in sodium pump number and activity in smooth muscle may underlie hypertension in preeclampsia. STUDY DESIGN: Northern and Western analyses were used to determine whether sodium pump alpha-isoform regulation in myometrium, placenta, and umbilical artery of women with preeclampsia differed from those with normotensive pregnancies. RESULTS: Levels of alpha1 and alpha3 messenger ribonucleic acid were reduced in myometrium of women with preeclampsia compared with normotensive pregnancies, as was alpha2 messenger ribonucleic acid in preeclamptic placenta. Protein expression of the alpha-isoforms was unaltered in placenta and umbilical artery from women with preeclampsia versus those with normotensive pregnancies, but myometrial alpha2 protein levels were reduced significantly in women with preeclampsia. Moreover, myometrial alpha1 protein expression was undetectable. CONCLUSIONS: Reduced smooth muscle sodium pump expression in preeclampsia may raise cell sodium, increase pressor sensitivity, or increase tone directly, which may contribute to hypertension in preeclampsia.

A labile sodium pump inhibitor from the peritoneal dialysate of hypertensive renal failure patients: estimates of potency.

Estimations of concentration of the labile sodium pump inhibitor isolated from human peritoneal dialysate were made using supercritical fluid chromatography coupled to flame ionization detection to determine the quantity of this factor in half of a purified preparation of the factor compared to the bioactivity of the other half in different assays. Ouabain was used for comparison. The labile factor appeared to be 30 times more effective than ouabain against canine renal [Na,K]ATPase. Moreover, this same factor appeared to be approximately 1,000 times more potent than ouabain in causing vascular smooth muscle contraction. The differences between this labile sodium pump inhibitor and ouabain most likely reflect their respective binding affinities. The assay differences in half maximal response to the labile sodium pump inhibitor may be due to differences in sodium pump alpha isoform sensitivity.

Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase.

Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases; moreover, the low selectivity of the mitochondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity. The purified enzyme demonstrated both polymerase and 3'-5' exonuclease activity. The purified protein was examined in single nucleotide incorporation assays, demonstrating that the enzyme had a maximum polymerization rate of 3.5 s-1 and a dissociation rate from the DNA substrate of 0.03 s-1, affording a calculated processivity of 116. The dissociation constants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 microM, respectively. The 3'-5' exonuclease rate was measured at 0. 18 s-1. Though the slow rate of polymerization suggests that the large subunit of human DNA polymerase gamma may require accessory factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma could replicate the mitochondrial genome in a physiologically relevant time frame. This study provides the initial characterization of the large subunit of DNA polymerase gamma and establishes the baseline for examination of the effects of accessory proteins such as the putative small subunit.

Tissue-specific regulation of the sodium pump in DOCA-salt hypertension.

Alterations in sodium pump activity have been associated with volume-sensitive hypertension, but little is known regarding the molecular regulation of the catalytically active alpha-subunit of the sodium pump in these models. We examined changes in the mRNA abundance of the alpha-isoforms in tissues that might participate in sodium and volume regulation in the deoxycorticosterone acetate (DOCA)-high salt rat model. These tissues included kidney, heart, aorta, pituitary, and hypothalamus. This study assessed alterations arising from changes in dietary salt intake alone, from DOCA administration alone, and those requiring both DOCA and high salt with their attendant volume expansion and hypertension. Increased sodium intake produced no significant change in any isoform in the five tissues studied. DOCA administered with a low salt diet produced no significant change in any of the alpha-isoforms in any of the tissues studied. The combination of DOCA and high salt (HS), on the other hand, brought about a twofold increase in renal alpha1-mRNA abundance compared with control (alpha1, CTL: 101.1 +/- 9.3, DOCA-HS: 197.3 +/- 22.9, P < .0001). DOCA-HS also induced a marked increase in both alpha1- and alpha2-mRNA in the aorta (alpha1, CTL: 122.5 +/- 33.3 v DOCA-HS: 487.2 +/- 59.9, P = .001; alpha2, CTL: 126.6 +/- 40.0 v DOCA-HS: 559.8 +/- 271.7, P = .01). In contrast DOCA-HS animals showed a significant reduction in the alpha2-, but not alpha1-, mRNA abundance in heart (alpha2, CTL: 118.0 +/- 11.7 v DOCA-HS: 61.1 +/- 9.1, P = .006). No change was observed in pituitary or hypothalamus with DOCA-HS. Of factors known to modulate the mRNA abundance of the sodium pump, only the putative endogenous sodium pump inhibitor might account for the changes in the aorta and kidney. Reductions in aldosterone or hypertension might reduce alpha2 in the heart. Only the renal response would favor sodium reabsorption, which could contribute to the hypertensive process.

Digoxin-like immunoreactive substance and sodium-potassium-adenosine triphosphatase inhibition in normal pregnancy: a longitudinal study.

OBJECTIVE: To measure the levels of digoxin-like immunoreactive substance and digitalis-like factor bioactivity as manifested by sodium-potassium-adenosine triphosphatase (ATPase) inhibition throughout pregnancy. METHODS: Serum samples were collected from primigravidas in early (15 +/- 1.8 weeks), mid (26 +/- 1.2 weeks), and late (36 +/- 1.1 weeks) gestation, as well as at 6 +/- 1.1 weeks postpartum (mean +/- standard error). Digoxin-like immunoreactive substance levels were determined by radioimmunoassay and digitalis-like factor bioactivity was determined by inhibition of ATPase. Data were analyzed by means of repeated measures analysis of variance. RESULTS: In 41 women with normal pregnancy outcomes, levels of digoxin-like immunoreactive substance rose progressively and significantly (P < .001) throughout pregnancy and returned to normal levels postpartum. Inhibition of ATPase activity also rose significantly (P < .004), but not as dramatically, during pregnancy and remained elevated 6 weeks postpartum. CONCLUSION: Although digoxin-like immunoreactive substance levels rise in pregnancy, functional digitalis-like factor activity, as manifested by inhibition of ATPase, does not parallel this rise strictly, implying that digoxin-like immunoreactive substance receptors may be reset during normal pregnancy. The enhanced cardiac performance that occurs in normal pregnancy may be mediated in part by increased digitalis-like factor activity.

Renal sodium pump regulation in deoxycorticosterone salt hypertension in the rat.

The renal sodium pump participates in sodium homeostasis and has been predicted to have a role in salt dependent forms of hypertension. However, the status of the renal sodium pump in volume-dependent hypertension is unclear. We assessed the renal sodium pump and its activity in the deoxycorticosterone acetate (DOCA)-salt model in rats, a model of volume-dependent hypertension. Sprague-Dawley rats on ad libitum diet were compared with four groups of litter mates receiving high or low salt diets, with or without DOCA administration. The renal sodium pump evaluation included measurement of hydrolytic activity, ouabain binding capacity and affinity, sodium activation, active pump units (determined by phosphoenzyme level), dephosphorylation rate, and isoform specific molecular expression. Intrinsic enzyme properties, including sodium and ouabain affinities, as well as turnover rate per sodium pump, were identical among the five groups. In contrast, the combination of DOCA and high salt intake (DOCA high salt) produced marked, significant increases in hydrolytic activity, ouabain binding capacity, phosphoenzyme level, and alpha1-isoform expression. DOCA low salt animals showed much smaller but significant increases in pump number. We conclude that DOCA and volume expansion may each alter renal sodium pump regulation, but volume expansion is clearly dominant. Increased renal sodium pump activity in DOCA high salt animals would, if unmitigated, favor sodium reabsorption and may contribute to hypertension.