The Federal University of São João del-Rei in association with the University of São Paulo, Federal University of Rio de Janeiro, Federal University of Goiás and Federal University of Paraíba has the honor to invite the scientific community to the VI Meeting on Cardiotonic Steroids and the Na+ Pump.
Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low-frequency Ca2+ oscillations. This ouabain mediated signal has within a couple of minutes resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin-regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process, regulation of cell adhesion and cell proliferation. Further evaluation of the physiological and pathophysiological consequences of the Na,K-ATPase signal emerges as an urgent topic for future studies.
Presented by Dr. Paula Kinoshita
Presented by Dr. Ana Maria Orellana
The renal proximal tubule (RPT) absorbs two-third of Na+ in the kidney and regulates systemic volume homeostasis. It has long been thought that Na/K-ATPase (NKA) functions primarily as an enzymatic ion-pump driving Na+ absorption in the RPT, promoting anti-natriuresis. More recently, it became apparent that RPT NKA forms a non-enzymatic receptor complex with Src-kinase. upon activation by cardiotonic steroids, this mechanism was shown to downregulate Na+ absorption in cultured RPT cells through coupled downregulation of NKA and NHE3 membrane abundance.
To establish the relative importance of these two functions of RPT NKA on Na+ and water homeostasis, we examined the effect of genetic downregulation of NKA expression in RPT in vitro and in vivo. Silencing NKA in RPT cell monolayers resulted in a doubling of cellular Na+ transport, without increase in paracellular transport. Genetic suppression of RPT NKA in mice increased renal Na+ absorption and markedly decreased daily urinary Na+ and water excretion. These effects were attributable to the dismantling of the NKA-Src signaling axis, through uncoupling and redistribution of Src, leading to unregulated plasmalemmal ion transporters. These studies suggest that RPT NKA signaling is not only physiologically important, it is also functionally dominant over the counteracting classical ion-pumping in the control of RPT Na+ absorption.
Presented by Dr. François Noël
We have previously demonstrated that the Na/K-ATPase signaling-mediated oxidant amplification loop contributes to experimental uremic cardiomyopathy and anemia induced by 5/6th partial nephrectomy (PNx). This process can be ameliorated by systemic administration of pNaKtide, which was designed to block this oxidant amplification loop. The present study demonstrated that the PNx induced anemia is characterized by marked decrease in RBC lifespan assessed by biotinylated RBC clearance and increase in RBC eryptosis as assessed by annexin V binding. No significant changes in plasma iron homeostasis were observed. Examination of plasma samples demonstrated that PNx induced significant systemic oxidant stress as assessed by protein carbonylation. Systemic administration of pNaKtide led to normalization of the oxidant stress, RBC lifespan and hematocrit without notable changes in iron metabolism. In preparation of RBC membrane fraction, we found Na/K-ATPase α1 subunit, c-Src, and caveolin-1, making the Na/K-ATPase signaling possible within RBCs. These data indicate that blockage of Na/K-ATPase signaling-mediated oxidant amplification loop ameliorates the anemia of experimental renal failure by increasing RBC lifespan and decrease in eryptosis.
Presented by Dr. Cristóforo Scavone
Presented by Dr. Elisa Kawamoto
Despite advances in chronic kidney disease (CKD) management strategies, patients with CKD often develop oxidative stress and a pro-inflammatory state. Inflammation and oxidant stress play a central role in the onset and progression of renal injury in CKD by inducing the release of cytokines and increasing expression of adhesion molecules, which together contribute to recruitment of more inflammatory cells to the kidney, subsequently promoting a pro-fibrotic milieu. We have demonstrated that cardiotonic steroids (CTS) are significantly elevated in CKD and are important mediators of inflammation and oxidative stress in this setting. CTS enhance renal inflammation and fibrosis upon binding and signaling through the Na+/K+-ATPase, and chronically elevated levels of CTS also have deleterious effects on the progression of renal and cardiovascular disease.
Paraoxonases are hydrolytic lactonase enzymes which are synthesized by the liver and circulate bound to high density lipoproteins (HDL). Our experimental and clinical data demonstrate an association between diminished lactonase activities of circulating PONs and progression of CKD. We have observed that renal inflammation and fibrosis are significantly increased following renal insult in animal models in which the circulating isoforms (PON-1 and PON-3) are knocked-out. We have also established that CTS-induced renal inflammation following renal insult is regulated by PONs. We show that PONsmodulate the CTS-Na+/K+-ATPase signaling axis and reduces the progression of renal inflammation, and renal impairment following renal insult. Taken together, our data suggest that PONs can attenuate the progression of renal inflammation in CKD and that the underlying mechanism involves attenuating the pathogenetic pathways induced by the CTS-Na+/K+-ATPase signaling axis. Our studies have shown for the first time a novel endogenous counter-regulatory mechanism of the CTS-Na+/K+-ATPase signaling activity which can be therapeutically targeted to attenuate inflammation and oxidant stress underlying renal impairment in CKD.
Presented by Dr. Sandra Mascarenhas
Na+/K+-ATPase is an ion pump and triggers a signaling pathway with diverse cellular effects. Leptospirosis causes hydro electrolytic disturbances. Our group unveiled the Na+/K+-ATPase as a molecular target of leptospiral glicolipoprotein (GLP). We showed the lipid fraction-containing oleic acid (OA) caused that inhibition. GLP, OA, and the cardiac glycoside ouabain induced experimental lung injury similar to the acute respiratory distress syndrome (ARDS) observed in critically ill patients. The release of GLP-containing OA targets tissues and impars membrane functions which may explain pathological changes characterizing the leptospiral infection. To assess enzyme activity we performed in vivo and in situ experiments. Patients with increased levels of unesterified fatty acids as OA have a poor prognosis, which at least partially may be explained by Na+/K+-ATPase inhibition. Activating Na+/K+-ATPase intracellular pathways triggers cell death, turning the enzyme into a promising target for cancer therapy. We showed perillyl alcohol (POH) caused Na+/K+-ATPase glioblastoma cell death. Other compounds, such as sesquiterpene polygodial and its derivates, retained activity against apoptosis- and multidrug-resistant cancer cells by inhibiting Na+/K+-ATPase. In a viral infection, cardiac glycosides altered ionic concentration in hosts, which regulates the virus life-cycle. The dual function of Na+/K+-ATPase could be explored as a molecular target to bacterial, tumoral, and viral diseases.
Presented by Dr. Luis Eduardo Quintas
Among the various isoforms of the Na,K-ATPase (NKA), the α4 polypeptide (NKAα4) is the one with the most limited pattern of expression, being restricted to the testis. Within the testis, NKAα4 is found in male germ cells, where it is expressed after cell meiosis. NKAα4 shows the highest expression levels in the mid-piece of the sperm flagellum. Deletion of NKAα4 in mice results in complete infertility of male, but not female animals. Activity of NKAα4 is essential for maintaining sperm intracellular Na+ levels and for the secondary control of several other vital sperm parameters, including membrane potential, intracellular Ca2+, and pH. Importantly, NKAα4 is crucial for sperm motility and sperm hyperactivation, a key event associated with sperm capacitation. These results support the biological relevance of NKAα4 in male fertility and its suitability as a pharmacological target for male contraception. From a biochemical perspective, and different from all other NKA α isoforms, NKAα4 has a distinct high sensitivity for the cardenolide ouabain. We have developed a series of ouabain-derivatives, in which the aglycone and lactone ring domains of the cardenolides were modified. Three of these compounds, SS-I-24, SS-I-42 and SS-I-54, exhibited a strong inhibitory activity for NKAα4 that was higher than that caused by ouabain. In addition, SS-I-24, SS-I-42 and SS-I-54 had a preferential effect on NKAα4, over the somatic Na,K-ATPase α1, α2 and α3 isoforms. Importantly, SS-I-24, SS-I-42 and SS-I-54 inhibited total and most aspects of sperm motility. In agreement with its IC50 inhibition value, SS-I-54 displayed the highest effect. SS-I-54 produced sperm plasma membrane depolarization, acidification and increased sperm [Ca2+]i, agreeing with the notion that SS-I-54 specifically targets NKAα4 function. Administration of SS-I-54 to rats via intraperitoneal (i.p.) injection, at concentrations as low as 2.5 mg/Kg weight, or through oral gavage, using a constant dose of 5 mg/Kg for different days (3, 6, 9 and 12 days), or a variable dose of 5, 10 and 20 mg/Kg for 3 days, inhibited sperm motility. Altogether, these results show that the synthetic cardenolides that we have generated are effective, selective inhibitors of NKAα4 activity and sperm motility both in vitro and ex vivo, supporting their use as male contraceptive agents. [Supported by NIH grant HD080423].
Presented by Dr. Leandro Barbosa
All biology depends first upon chemistry. The mammalian brain, especially that of humans, is the most sophisticated biological structure known to man utilizing a massive array of chemical entities. Steroids are a crucial class of chemical entities within the CNS. They impact numerous functions including mood, sexual orientation, memory, blood pressure, salt and water balance, etc. Included among these steroids are endogenous cardiotonic steroids (CTS) including ouabain – ordinarily considered to be a CTS of plant origin. Ouabain and its endogenous mammalian counterpart (EO) are highly polar CTS that are structurally identical but with different origins. Under normal conditions (intact blood brain barrier), neither has free access to the CNS. However, both can enter the hypothalamus in limited amounts via the fenestrated epithelia surrounding the circumventricular organs of the hypothalamus. This region is heavily invested in the control of water and electrolyte balance, sympathetic nerve activity and long-term blood pressure control. Within the CNS, EO-like materials are highly enriched in discrete hypothalamic neurons that project from the supraoptic and paraventricular nuclei to the rostral ventral medulla and pituitary. The hypothalamic (and pituitary) content of EO represents a mix of local hypothalamic synthesis plus that coming via the circulation from the adrenal glands (Hamlyn et al, 2014, Leenen et al, 2020). Elsewhere in the CNS, under normal circumstances, most of the CNS EO arises from local synthesis. Within the brain, there are polymorphic receptors for EO as reflected by the sodium pump α subunit isoforms. But are there polymorphic ligands for these sodium pump isoforms? Many classic mammalian steroids have numerous isomeric analogs but, thus far, no naturally occurring isomers of ouabain in the plant kingdom have been described. Are there isomers of EO in mammals and if so where are they and what do they do? Some answers to these questions arose by accident during an investigation of the effects of CNS angiotensin II (Ang II) on long-term blood pressure in rodents. It is well accepted that sustained increases in CNS Ang II stimulate angiotensin II type I receptors (AT1R), activate the sympathetic nervous system and cause hypertension. Remarkably, the mechanism by which this occurs involves aldosterone and EO synthesis in the CNS. Blockade of aldosterone receptors as well inhibition of aldosterone synthesis in the brain prevents hypertension and returns the elevated circulating EO (measured by routine radioimmunoassay (RIA)) to normal. When the RIA measurements were repeated after HPLC to confirm that EO per se was the basis of the immunocrossreactivity, two immunoreactive peaks in addition to EO were detected. These were proven subsequently to be EO isomers by multidimensional mass spectrometry. Isomer 1 was more polar than EO and minimally impacted by CNS blockade of aldosterone. In contrast, isomer 2 was less polar than EO, was sensitive to CNS Ang II and aldosterone receptor blockade, but was increased >30 fold in the circulation of animals in which the brain synthesis of aldosterone was suppressed by fadrazole. Thus, this work demonstrates that the circulation of rodents contains multiple ligands patterned on EO that, together with multiple Na pump isoforms, add to the already wide biological panorama in this arena. In contrast to the periphery, the CNS is a privileged space surrounded by a blood brain barrier. In the CNS large chemical gradients can occur as the result of local biosynthesis. To determine whether EO and these isomers are present in discrete regions in the CNS, fresh adult bovine brains were interrogated by direct electrospray mass spectrometry. EO and isomer 2 were found throughout the CNS but not always together. Moreover, five discrete “hot areas” were observed for EO where its concentrations were 10-100x higher than the surrounding tissue. Two hot areas were found for isomer 2, only one of which overlapped with EO. Hot areas were especially prominent in the hypothalamus, brain stem and the hippocampus. The observation that these isomers are present in rodents and cattle suggests they are of widespread significance in mammals and likely play a targeted role in Na pump regulation and signaling especially in discreet brain regions. Several of these EO enriched areas are in locations relevant to CNS conditions including mania and bipolar disorders, depression, epilepsy, and memory. All of these hot areas have been linked previously with both beneficial as well as adverse dose-dependent actions of EO, ouabain and other CTS. Further, the hypothalamic and brain stem sites have obvious and direct relevance to the long term hypertensinogenic actions of ouabain and EO already documented by many studies. The interactions of isoforms and isomers and their impact on biology in health and disease represent a new chapter in the book of biology.
Presented by Dr. Luciana Rossoni
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