Ulrike Kirch

Bovine Adrenals Contain, in Addition to Ouabain, a Second Inhibitor of the Sodium Pump

In the search for endogenous cardiac glycosides, two different inhibitors of the sodium pump have been isolated from bovine adrenals. Inhibitor A with a molecular mass of 600 Da and a UV maximum at 250 nm was purified from 16 kg of bovine adrenals. The pure substance (<1 ng) inhibited the sodium pump of human red blood cells with an affinity similar to that of ouabain, yet it cross-reacted with antibodies against the bufadienolide proscillaridin A but not against the cardenolide ouabain. Inhibitor A was slightly more hydrophilic than ouabain on RP-C18 high pressure liquid chromatography. Hence, it showed properties similar to the proscillaridin A immunoreactivity (Sich, B., Kirch, U., Tepel, M., Zideck, W., and Schoner, W. (1996) Hypertension 27, 1073–1078) that increased in humans with systolic blood pressure and pulse pressure. Inhibitor B of the sodium pump with a molecular mass of 584 Da was purified 106-fold from 20 kg of bovine adrenals. It cross-reacted with antibodies against ouabain but not with antibodies against proscillaridin A and inhibited the sodium pump of human and rat red blood cells with the same affinity as ouabain. All other properties, such as the retention time in a C18-reversed phase chromatography, molecular mass determination by electrospray mass spectrometry and fragmentation pattern, and UV and 1H NMR spectroscopic data, were identical to ouabain. Hence, sodium pump inhibitor B from bovine adrenals is the cardenolide ouabain.

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs: Effects of β-Blockade and Angiotensin-Converting Enzyme Inhibition

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by 86Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5±0.5 to 86.0±27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3±1.7 to 129.8±51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1±1.2 nmol/L and at 30±1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7±0.8 to 166.9±91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the &bgr;1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

Pulse pressure correlates in humans with a proscillaridin A immunoreactive compound

Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.

Ouabain as a Mammalian Hormone

Abstract: Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the μ chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na+,K+‐ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.

Dehydroepiandrosterone sulfate mediates activation of transcription factors CREB and ATF-1 via a Gα11-coupled receptor in the spermatogenic cell line GC-2.

Dehydroepiandrosterone sulfate (DHEAS) is a circulating steroid produced in the adrenal cortex, brain, and gonads. Whereas a series of investigations attest to neuroprotective effects of the steroid in the brain, surprisingly little is known about the physiological effects of DHEAS on cells of the reproductive system. Here we demonstrate that DHEAS acting on the spermatogenic cell line GC-2 induces a time- and concentration-dependent phosphorylation of c-Src and Erk1/2 and activates the transcription factors activating transforming factor-1 (ATF-1) and cyclic AMP-responsive element binding protein (CREB). These actions are consistent with the non-classical signaling pathway of testosterone and suggest that DHEAS is a pro-androgen that is converted into testosterone in order to exert its biological activity. The fact, however, that steroid sulfatase mRNA was not detected in the GC-2 cells and the clear demonstration of DHEAS-induced activation of Erk1/2, ATF-1 and CREB after silencing the androgen receptor by small interfering RNA (siRNA) clearly contradict this assumption and make it appear unlikely that DHEAS has to be converted in the cytosol into a different steroid in order to activate the kinases and transcription factors mentioned. Instead, it is likely that the DHEAS-induced signaling is mediated through the interaction of the steroid with a membrane-bound G-protein-coupled receptor, since silencing of Guanine nucleotide-binding protein subunit alpha-11 (Gnα11) leads to the abolition of the DHEAS-induced stimulation of Erk1/2, ATF-1, and CREB. The investigation presented here shows a hormone-like activity of DHEAS on a spermatogenic cell line. Since DHEAS is produced in male and female reproductive organs, these findings could help to define new roles for DHEAS in the physiology of reproduction.

Proscillaridin A Immunoreactivity: Its Purification, Transport in Blood By A Specific Binding Protein And Its Correlation With Blood Pressure

A material crossreacting with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump was found in human blood plasma. The concentration of the material with a retention time similar to ouabain in a reversed phase HPLC correlated to systolic blood pressure and pulse pressure. Affinity purification of this compound from bovine adrenals resulted in the isolation of a compound with molecular mass of 600 Da that was not identical with ouabain. Consistent with the postulate that endogenous ouabain and proscillaridin A immunoreactivities may belong to a new class of cardiotonic steroid hormones, a protein of Mr = 60 kDa has been found in bovine serum by affinity-labeling with N-hydroxysuccimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate.

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Interaction of the α2A domain of integrin with small collagen fragments

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.

Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility.