Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid-Induced Increase in Heart Contractility.

Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.

Endogenous ouabain regulates cell viability.

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.

The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors.

RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation.

RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains.

Members of the RIC-3 gene family are effectors of nicotinic acetylcholine receptor (nAChR) expression in vertebrates and invertebrates. In Caenorhabditis elegans RIC-3 is needed for functional expression of multiple nAChRs, including the DEG-3/DES-2 nAChR. Effects of RIC-3 on DEG-3/DES-2 functional expression are found in vivo and following heterologous expression in Xenopus leavis oocytes. We now show that in X. leavis oocytes RIC-3 also affects the kinetics and agonist affinity properties of the DEG-3/DES-2 receptor. Because these effects are mimicked by increasing the ratio of DEG-3 subunits within DEG-3/DES-2 receptors, this suggests that RIC-3 may preferentially promote maturation of DEG-3-rich receptors. Indeed, effects of RIC-3 on functional expression of DEG-3/DES-2 positively correlate with the DEG-3 to DES-2 ratio. All RIC-3 family members have two transmembrane domains followed by one or two coiled-coil domains. Here we show that the effects of RIC-3 on functional expression and on receptor properties are mediated by the transmembrane domains and do not require the coiled-coil domains. In agreement with this, mammals express a RIC-3 transcript lacking the coiled-coil domain that is capable of promoting DEG-3/DES-2 functional expression. Last, we show that RIC-3 affects DEG-3 quantity, suggesting stabilization of receptors or receptor intermediates by RIC-3. Together our results suggest that subunit-specific interactions of RIC-3 with nAChR subunits, mediated by the transmembrane domains, are sufficient for the effects of RIC-3 on nAChR quantity and quality.