Garner T. Haupert

Endogenous Ouabain Upregulation of Steroidogenic Genes in Hypertensive Hypothalamus but Not Adrenal

Background—Mammalian tissues contain a presumed endogenous Na+, K+-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. Methods and Results—Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and &Dgr;5-3&bgr;-hydroxysteroid dehydrogenase/&Dgr;5-&Dgr;4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. Conclusions—Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

Hypoxia triggers release of an endogenous inhibitor of Na+-K+-ATPase from midbrain and adrenal

An endogenous inhibitor of Na+-K+-ATPase has been isolated from bovine hypothalamus and human plasma and structurally characterized as an isomer of the plant cardiac glycoside, ouabain (A. A. Tymiak, J. A. Norman, M. Bolgar, G. C. DiDonato, H. Lee, W. L. Parker, L.-C. Lo, N. Berova, K. Nakanishi, E. Haber, and G. T. Haupert, Jr. Proc. Natl. Acad. Sci. USA 90: 8189-8193, 1993; N. Zhao, L.-C. Lo, N. Berova, K. Nakanishi, J. H. Ludens, and G. T. Haupert, Jr. Biochemistry 34: 9893-9896, 1995). This hypothalamic inhibitory factor (HIF) acts on cardiovascular and renal tissues consistent with physiological regulation in vivo. Stimuli for the release of HIF from tissue are unknown. Hypoxia may be a stimulus for the elaboration of digitalis-like activity in humans, and high NaCl concentration in central nervous system stimulates ouabain-like activity in animals. We examined the ability of low O2 tension in vivo and in vitro to stimulate HIF release from midbrain and adrenal tissues in Wistar rats. In both tissues, hypoxia stimulated a remarkable release of an inhibitor cochromatographing with HIF, and this release was enhanced by 300 mM NaCl. Plasma from hypoxic rats also showed increased levels of the purified inhibitory activity. We conclude that hypoxia is a potent stimulus for the release of HIF or HIF-like activity and discuss the possibility that an Na+-K+-ATPase inhibitor could be involved in energy-conserving cellular adaptive responses to hypoxic or ischemic insult through ATP conservation.

Hypothalamic Na+,K(+)-ATPase inhibitor constricts pulmonary arteries of spontaneously hypertensive rats.

Hypothalamic inhibitory factor (HIF) is an endogenous high-affinity inhibitor of Na+,K(+)-ATPase with ouabain-like properties and has been implicated in the pathogenesis of genetic systemic hypertension. We wondered whether HIF might also be associated with the recently demonstrated pulmonary hypertension of spontaneously hypertensive rats (SHRs). We compared HIF effects on the contractility of isolated 2- to 3-mm pulmonary artery (PA) rings from SHRs and age-matched normotensive Sprague-Dawley (SD) rats. HIF caused a reversible, concentration-dependent increase in tension in PA rings of SHR and SD rats, whereas ouabain did not. PA tension development with HIF (4 nM final concentration) was significantly higher in SHRs than in SD rats: 308 +/- 56 mg (mean +/- SE) vs. 137 +/- 26, respectively, p < 0.05. Abdominal aortic contractions induced by HIF did not differ between SHRs and SD rats. In SHRs, but not SD rats, the effect on PA rings was significantly greater than on aortic rings. In all cases, contraction was abolished by phentolamine but was unaffected by calcium-channel blockade using verapamil. HIF-induced tension development required external Ca2+. We conclude that PA rings from SHRs are more sensitive to Na+,K(+)-ATPase inhibitory effects of HIF than PA rings from SD rats, which may contribute to the observed pulmonary hypertension in SHR. Local modulation of the Na+,K(+)-ATPase-adrenergic neuroeffector interaction may be the vasoconstrictive mechanism of action of HIF in these vessels.

Ouabain-Binding Protein(s) From Human Plasma

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18 inhibit Na+ pump activity in human erythrocytes (IC50≈4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (≤0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.

Erythrocyte sodium/potassium ATPase activity in severe preeclampsia.

Objective:Elevated blood levels of endogenous digitalis-like factors (EDLF) may decrease erythrocyte sodium pump activity in preeclampsia. As the highest EDLF levels might be expected in severe preeclampsia, we investigated sodium pump activity in that group of patients.Study Design:Erythrocyte sodium pump activity was determined by 86Rubidium uptake (in nM per hour per 106 cells) in women with severe preeclampsia and those with normal pregnancies, matched for gestational age, and in healthy nonpregnant women (n=12 in each group). Differences between groups were analyzed by a two-sided Student t-test.Result:Sodium pump activity was significantly increased in normotensive pregnancies as compared with normotensive non-pregnant women (81.4±8.4 vs 61.1±7.4, mean±s.d., p<0.05), and was decreased 43% in severe preeclamptic pregnancies as compared with normotensive pregnancies (46.4±14.1 vs 81.4±8.4, p<0.05).Conclusion:Severe preeclampsia is associated with significantly lower erythrocyte sodim pump activity than normotensive pregnancy. These data suggest that plasma levels of a biologically active EDLF are elevated in patients with severe preeclampsia.

Use of Two-Sided Bifunctional Liposomes in the Study of a Hypothalamic Na,K-ATPase Inhibitor

Two-sided bifunctional (ATP-filled) Na,K-ATPase liposomes have been developed as a result of knowledge about the average liposome diameter and volume, the liposome size distribution, the average number of Na,K-ATPase molecules reconstituted per liposome, and the orientation of the reconstituted Na,K-ATPase molecules. The addition of 5-10 microM external 86Rb to the liposomes containing 50 mM encapsulated ATP provoked an impressive 86Rb accumulation by the cell-like-oriented pumps. The successive addition of external ATP activated the pumps in the reversed orientation of the same liposome, leading to total extrusion of the previously accumulated 86Rb. An inhibitor extracted from bovine hypothalamus (hypothalamic inhibitory factor) inhibited the cell-like-oriented population, i.e., acted like an extracellular inhibitor at 30 nM. Conversely, at 75 nM, the reversed pump population was also blocked, indicating that the inhibitor either transversed the membrane or was able to act also at the intracellular enzyme side at a higher concentration. Thus, the side of action as well as the membrane permeability of structurally unknown endogenous Na,K-ATPase inhibitors can be determined simultaneously in a single suspension of two-sided bifunctional Na,K-ATPase liposomes.

Search for an endogenous mammalian cardiotonic factor.

A number of cardiotonic factors have been characterized from the plants Digitalis purpurea, Strophanthus gratus, etc., and from a variety of toads Bufo.1,2 The genin of the plant factors are polyhydroxylated C17-steroids with an α,β-unsaturated γ-lactone attached to C-17 (β) and various sugars attached to C-3 (Fig. 1). The genin of the toad factors (C18 steroids) contains an α-pyrone at C-17 instead of the γ-lactone.

Circulating inhibitors of sodium transport at the prehypertensive stage of essential hypertension.

Summary: Alterations in cellular calcium metabolism are presumed to he the basis of the vasoconstrietive process that sustains elevations in arterial pressure. Altered membrane sodium transport can effect changes in intracellular flee calcium concentration through changes in transmembrane sodium gradients and membrane depolarization. Thus, changes in membrane sodium transport could produce calcium accumulation in vascular smooth muscle, resulting in vasoconstriction and arterial hypertension. Multiple sodium transport abnormalities exist in tissues of genetically hypertensive rats, and blood cells of human essential hypertensive. Na -K cotransport and Na -Li countertransport abnormalities appear to he primary membrane defects, but a direct physiologic link of these to vasoconstriction remains to he established. Evidence for circulating Na.K–ATP axe inhibitors in hypertension is now widely reported, and Na.K–ATPase inhibition provides a rationale for vasoconstriction through altered calcium and or neurotransmitter metabolism. Na.K–ATPase inhibition in hypertensive disease appears to arise not as a primary abnormality in membrane transport, nor as a phenomenon secondary to hypertension per se. but as a physiological response to compensate for excess extracellular fluid volume accumulation.

Measurement of glomerular filtration rate in anesthetized and conscious rhesus monkeys (Macaca mulatta).

OBJECTIVE To validate a method to assess glomerular filtration rate (GFR) in conscious monkeys via transcutaneous radiation detection after IV injection of technetium Tc 99m pentatate (99mTc-DTPA). ANIMALS 4 healthy rhesus monkeys. PROCEDURES On day 1, each monkey was anesthetized, lothalamate sodium I 125 (125l-iothalamate) was administered via continuous rate infusion (0.0037 MBq/min); blood and urine samples were obtained for determination of 125l-iothalamate plasma clearance variables and estimation of GFR. One dose of 99mTc-DTPA (74 MBq/kg, IV) was also administered during the 125l-iothalamate plasma clearance test, and transcutaneous measurements of technetium 99m-emitted radiation were obtained by use of an ambulatory renal monitor (ARM) applied to a brachium of each monkey. Determination of GFR by use of the ARM was repeated on days 8 and 45 in the same monkeys without anesthesia. RESULTS Sensitivity, accuracy, and precision of the 2 methods were similar. By use of the ARM, GFR determined by use of the renal rate constant (κGFR) was calculated; the value obtained on day 1 under anesthesia was similar to values determined via 125l-iothalamate plasma clearance testing on the same day, but was 16% to 23% less than that measured on days 8 and 45 in conscious monkeys. CONCLUSIONS AND CLINICAL RELEVANCE The ARM method for assessment of GFR was less invasive, faster, and more convenient than the standard clearance method, but yielded comparable results. The need to train animals and size restrictions of the device may limit the use of this technique in other nonhuman animals.

89 Metabolism of uridine in expanded extracellular volume states

1. Uridine and uridine monophosphate (UMP) are natriuretic and a vasopressor in intact rats. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats metabolic clearance rate (MCR) of uridine is raised and basal plasma uridine diminished, suggesting that metabolism of uridine is linked to changes in extracellular space. 2. Plasma uridine concentration was raised in 38 patients with chronic renal failure compared with age- and sex-matched healthy controls (8.49 mumol/L, 4.37-13.74 mumol/L median, interquartile range, and 2.64 mumol/L 2.51-2.74 mumol/L, respectively, P < 0.001). Plasma uridine was significantly diminished after isotonic fluid removal by ultrafiltration (UF) from 7.25 mumol/L (3.7-11.08) to 5.07 mumol/L (3.3-8.3), P < 0.001, whereas concentration of marker solutes urea and creatinine remained unchanged. During haemodialysis (HD), plasma uridine fell significantly from its pre-HD level. 3. In an animal model of expanded extracellular space the one-kidney, one-clip rat, plasma uridine was significantly higher (20.56 +/- 1.19 mumol/L, P < 0.01) and MCR diminished (34.93 +/- 3.44 mL/kg per min, P < 0.01) compared with sham-operated animals (plasma uridine 12.14 +/- 1.07 and MCR 53.59 +/- 4.11 mL/kg per min). Uridine or UMP did not inhibit Na+, K(+)-ATPase in either of the two assay systems. 4. It was concluded that catabolism of uridine is reduced by extracellular expansion and probably increased by volume reduction by UF.(ABSTRACT TRUNCATED AT 250 WORDS)