Renata I. Dmitrieva

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia

OBJECTIVE To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma. DESIGN AND METHODS Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries. RESULTS In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery. CONCLUSIONS Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Characterization of a Urinary Bufodienolide Na+,K+-ATPase Inhibitor in Patients After Acute Myocardial Infarction

Recent evidence suggests the existence of several endogenous Na+,K+-ATPase inhibitors in mammals. Previously, we have shown that the amphibian Na+,K+-ATPase inhibitor marinobufagenin (3,5-dihydroxy-14,15-epoxy bufodienolide) acts as a vasoconstrictor in isolated rat and human arteries. Mammalian plasma was shown to contain marinobufagenin-like immunoreactive material, which is responsive to saline volume expansion. The present study describes purification of a bufodienolide, which is similar to marinobufagenin, from the urine of patients after acute myocardial infarction with the use of thin-layer chromatography and reverse-phase high-performance liquid chromatography (HPLC). The purified substance cross-reacted with marinobufagenin antibody, demonstrated maximal UV absorbance at 300 nm characteristic of bufodienolides, and eluted from HPLC columns with the same retention time as marinobufagenin. Mass spectrometry of purified material revealed the presence of a substance indistinguishable from amphibian marinobufagenin and having molecular mass of 400 D. The present studies show that one of the human digitalis-like factors may have a bufodienolide structure and is likely to represent marinobufagenin or its isomer, and they suggest a role for this substance in the pathogenesis of myocardial ischemia.

Mammalian Bufadienolide Is Synthesized From Cholesterol in the Adrenal Cortex by a Pathway That Is Independent of Cholesterol Side-Chain Cleavage

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na+,K+-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.

Cardiotonic steroids: Potential endogenous sodium pump ligands with diverse function

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.

Protein kinase M zeta regulation of Na/K ATPase: a persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures.

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia-hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMzeta), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the alpha1 and alpha2 isoforms of the Na/K ATPase. Similarly, active PKMzeta levels were increased at 24 h, but not 2 h, after preconditioning. PKMzeta overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMzeta regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit alpha1, dihydroouabain to inhibit alpha2/3 and a zeta-pseudosubstrate peptide to inhibit PKMzeta. These experiments showed that PKMzeta regulated both the activity and surface expression of the alpha1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and zeta-pseudosubstrate peptide were used to determine if PKMzeta or the alpha1 and alpha2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMzeta levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMzeta and Na/K ATPase in ischemic preconditioning.

Cicletanine reverses vasoconstriction induced by the endogenous sodium pump ligand, marinobufagenin, via a protein kinase C dependent mechanism.

Rationale Cicletanine (CIC), an anti-hypertensive compound with direct vascular and natriuretic actions, is especially effective in salt-sensitive hypertension, in which dysregulation of the sodium pump plays an important pathogenic role, and digitalis-like cardiotonic steroids contribute to increased vascular tone. The purpose of the present study was to investigate whether, and by what mechanisms, cicletanine antagonizes the vasoconstrictor effects of cardiotonic steroids in isolated human arteries. Methods The effects of cicletanine on vascular tone were studied in isolated, endothelium-denuded rings of 2nd-3rd-order branches of human mesenteric arteries pre-contracted with bufodienolide marinobufagenin (MBG), an Na/K-ATPase inhibitor, or endothelin-1 (ET-1). Na/KATPase activity was measured in sarcolemmal membranes from the mesenteric artery. Activity of rat brain protein kinase C (PKC) was measured using the PepTag phosphorylation assay. Results MBG and ET-1 both induced sustained vasoconstriction in human mesenteric artery rings, and cicletanine relaxed rings pre-contracted with either MBG (EC50 =11 ± 2 μmol/l) or ET-1 (EC50 = 6.4 ± 1.1 μmol/l). Although 8-Br-cGMP (100 μmol/l) caused complete vasorelaxation of arterial rings pre-contracted with ET-1, it did not affect the MBG-induced vasoconstriction. An activator of PKC, phorbol diacetate (PDA) (50 nmol/l), attenuated CIC-induced vasorelaxation of mesenteric artery rings pre-contracted with MBG (EC50 >100 μmol/l), but not rings pre-contracted with ET-1 (EC50 = 6.5 ± 1.2 μmol/l). In mesenteric artery sarcolemma, 100 nmol/l MBG inhibited the Na/K-ATPase by 68 ± 5% and cicletanine (100 μmol/l) attenuated this Na/K-ATPase inhibition by 85 ± 6%. In the PepTag PKC assay, cicletanine produced a concentration-dependent inhibition of rat brain PKC activity (IC50 45 ± 11 μmol/l). In the presence of 50 nmol/l PDA, 100 μmol/l cicletanine did not antagonize the Na/K-ATPase inhibition by MBG, and did not inhibit the PKC from rat brain. Conclusions Cicletanine antagonizes vasoconstriction induced by Na/K-ATPase inhibition via a PKC-dependent mechanism that does not involve inhibition of cyclic GMP phosphodiesterase (cGMP-PDE). This mechanism of action may be relevant to the greater potency of cicletanine in salt-sensitive hypertension in which plasma levels of endogenous digitalis-like cardiotonic steroids are elevated. Our findings also suggest that PKC is an important factor for cardiotonic steroid–Na/K-ATPase interactions on the vascular tone, and is therefore a potential target for therapeutic intervention in hypertension.

Genome-Wide Identification of Allelic Expression in Hypertensive Rats

Background—Identification of genes involved in complex cardiovascular disease traits has proven challenging. Inbred animal models can facilitate genetic studies of disease traits. The spontaneously hypertensive rat (SHR) is an inbred model of hypertension that exists in several closely related but genetically distinct lines. Methods and Results—We used renal gene-expression profiling across 3 distinct SHR lines to identify genes that show different expression in SHR than in the genetically related normotensive control strain, Wistar-Kyoto. To ensure robust discovery of genes showing SHR-specific expression differences, we considered only those genes in which differential expression is replicated in multiple animals of each of multiple hypertensive rat lines at multiple time points during the ontogeny of hypertension. Mutation analysis was performed on the identified genes to uncover allelic variation. We identified those genes in which all SHR lines share a single allele of the gene when normotensive controls (Wistar-Kyoto) have fixed the alternative allele. We then identified which of the differentially expressed genes show expression that is controlled by the alleleic variation present in and around the gene. Allelic expression was demonstrated by observing the effect on gene expression of alleles inherited in the freely segregating F2 progeny of a cross between SHR and Wistar-Kyoto animals. Conclusions—The result of these studies is the identification of several genes (Ptprj, Ela1, Dapk-2, and Gstt2) in which each of 4 SHR lines examined have fixed the same allele and in which each of 2 Wistar-Kyoto lines have a contrasting allele for which the inherited allele influences the level of gene expression. We further show that alleles of these genes lie in extensive haplotype blocks that have been inherited identical by descent in the hypertensive lines.

Regulation of adrenocortical cardiotonic steroid production by dopamine and PKA signaling

There is growing evidence that the adrenal cortex is the source of cardiotonic steroid (CS) regulators of sodium, potassium-ATPase (NKA). The control of adrenocortical production CS may play a critical role in mediating renal and vascular responses involved in arterial hypertension. Dopamine (DA) controls renal NKA by direct regulatory phosphorylation and indirectly by modification of aldosterone release. In the present studies, Y-1 adrenocortical cell cultures which have been shown to produce a cardiotonic steroid indistinguishable from the known vertebrate steroid, marinobufagenin (MBG), were treated with various agents to stimulate or antagonize dopamine signaling pathways. We demonstrate that Y-1 cells express both pharmacological types of dopamine receptor (DA1 and DA2). Treatment of Y-1 cells with DA stimulated MBG production in a dose range similar to that shown to inhibit aldosterone production by the adrenal cortex. Experiments with specific DA1 and DA2 receptor agonists and antagonists were performed and allowed us to attribute the DA stimulatory effect to a DA1 type receptor. The DA stimulatory effect on MBG depended on protein kinase A (PKA) and could be blocked by Rp-cAMPS. Although both basal and forskolin-stimulated progesterone production by Y-1 cells were profoundly inhibited in Y-1 cell lines expressing the dominant negative type I regulatory subunit of PKA, both basal and forskolin-stimulated MBG production were demonstrated in these lines. This evidence suggests a possible role of DA1 signaling through camp-mediated activation of the type II PKA holoenzyme in the adrenal cortex.

Vasorelaxant effects of cicletanine and its (+)- and (−)-enantiomers in isolated human pulmonary arteries

The purpose of the study was to investigate, in isolated human pulmonary artery, the ability of cicletanine and its (-) and (+)-enantiomers to attenuate the endothelin-1 (Et-1) induced vasoconstriction, and to potentiate vasorelaxation (relative to plateau of the effect of Et-1) by sodium nitroprusside (SNP) and human atrial natriuretic peptide (ANP). In pulmonary artery rings, Et-1 induced a concentration-dependent vasoconstriction with median effective concentration (EC50 = 26+/-2.8 nmol/L. Pretreatment of the vessels with 100 micromol/L (+/-)-cicletanine reduced the effect of Et-1 (EC50 = 36+/-3.5 nmol/L; P < .01). (-)-enantiomer displayed greater capacity to antagonize the vasoconstrictor action of Et-1 (EC50 = 47+/-4.2 nmol/L) v (+)-enantiomer (EC50 = 29.9+/-6.5 nmol/L; P < .01). In arterial rings, precontracted with 10 nmol/L Et-1, ANP caused vasorelaxation (EC50 = 9.7+/-1.9 nmol/ L). The relaxant effect of ANP was potentiated by 100 micromol/L of (-)-(EC50 = 4.2+/-0.6 nmol/L; P < .01), but not (+)-cicletanine (EC50 = 7.6+/-0.7 nmol/L). Sodium nitroprusside relaxed pulmonary artery rings precontracted with 10 nmol/L Et-1 (EC50 = 41+/-11 nmol/L). The effect of SNP was potentiated by 10 micromol/L (+/-)-cicletanine (EC50 = 9.0+/-0.7 nmol/L; P < .05). The potentiating effect of 10 micromol/L (+)-cicletanine was weaker (EC50 = 7.9+/-1.8 nmol/L) than that of (-)-enantiomer (EC50 = 3.3+/-0.54 nmol/L; P < .05). The relaxant effect of SNP was not further potentiated by 100 micromol/L (+/-)-cicletanine. The present results demonstrate that, cicletanine antagonizes Et-1 induced vasoconstriction in an isolated human pulmonary artery and potentiates vasorelaxation by two guanylate cyclase activators, ANP and SNP. (-)-Cicletanine displays greater vasorelaxant activity v (+)-enantiomer.

Genetic control of serum marinobufagenin in the spontaneously hypertensive rat and the relationship to blood pressure

Background We have investigated serum levels of immunoreactive marinobufagenin (MBG) in 16‐ to 20‐week‐old spontaneously hypertensive rats (SHRs)‐A3 and in the normotensive Wistar‐Kyoto (WKY) rat strain in the absence of salt loading, and we have investigated the genetic control of serum MBG. Methods and Results We genotyped the F2 progeny of an SHR‐A3×WKY intercross using a genome‐wide panel of 253 single‐nucleotide polymorphism markers that were dimorphic between SHR‐A3 and WKY and measured serum MBG by ELISA. Serum MBG levels were lower in SHR‐A3 than WKY rats (0.39±0.07 and 1.27±0.40 nmol/L, respectively), suggesting that MBG may not play a role in the markedly divergent blood pressure measured by telemetry in rats of these 2 strains (SHR‐A3 and WKY, 198.3±4.43 and 116.8±1.51 mm Hg, respectively). The strain difference in serum MBG was investigated to determine whether genomic regions influencing MBG might be identified by genetic mapping. Quantitative trait locus mapping indicated a single locus influencing serum MBG in the region of chromosome 6q12. Homozygosity of WKY alleles at this locus was associated with increased serum MBG levels. We surveyed whole genome sequences from our SHR‐A3 and WKY lines, seeking coding sequence variation between SHR‐A3 and WKY within the mapped locus that might explain the inherited strain difference in serum MBG. Conclusions We identified amino acid substitution in the sterol transport protein Abcg5, present in SHR‐A3, but absent in WKY, that is a potential mechanism influencing MBG levels.