Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radio-immunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immuno-reactivity with authentic ouabain. In most subjects no ouabain immunoreactivity that coeluted with authentic ouabain was observed. Some subjects had ouabain-immunoreactive material present at low levels, but it was largely attributable to cross-reactivity with diverse substances found not to be oua-bain. Similar results were obtained after analysis of plasma collected from 10 patients entering a medical intensive care unit. Studies of serum-free medium conditioned by bovine adrenocortical cells showed some ouabain immunoreactivity. To determine whether this material might be a steroid product of cholesterol side-chain cleavage, we performed chemical blockade of steroidogenesis, which effectively suppressed pro-gesterone production by these cells but had no consistent effect on ouabain immunoreactivity in this medium. Stimula-N umerous studies have attempted to identify and evaluate potential endogenous regulators of sodium-potassium ATPase activity that function via the cardiac glycoside binding site on this enzyme. 13 Of the candidate materials identified, none met the necessary criteria of specificity, selectivity, effective concentration range in plasma, and evidence for physiologically meaningful alterations in plasma levels until recent studies indicating that ouabain, or a stereoisomer of ouabain, was an endogenous mammalian material, apparently derived from the adrenal cortex. 4-6 This observation is surprising for a variety of reasons. Ouabain previously has been known only as a plant-derived material. The sugar rhamnose, which is attached to the C3 position of the A ring of the ouabain steroid nucleus, has previously been considered a plant sugar, and there is only sparse evidence for its utilization in mammalian metabolism. 7 " 9 Furthermore, oua-79430. tion of steroidogenesis with 22-R-OH-cholesterol in bovine adrenocortical cells did not produce any increase in the ouabain immunoreactivity present in conditioned medium. Subsequent HPLC studies of ouabain immunoreactivity in bovine adrenocortical cell-conditioned medium indicated that authentic ouabain did not account for most of the ouabain immunoreactivity in serum-free medium. Studies with bovine adrenocortical cells incubated in a minimal salt and glucose medium indicated a small peak of immunoreactivity that may correspond to authentic ouabain. Examination of ouabain immunoreactivity in serum-supplemented medium conditioned by the murine adrenocortical tumor cell line Yl indicated much higher levels of immunoreactivity; however, the major portion of this immunoreactivity eluted during reversed-phase HPLC with ...
We used confocal Ca 2+ imaging and the patch-clamp technique to investigate the interplay between Ca2+ entries through L-type Ca 2+ channels (LCCs) and reverse-mode Na + -Ca 2+ exchange (NCX) in activating Ca 2+ -induced Ca 2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N (6),2 -O-dibutyryl adenosine-3 ,5 -cyclic monophosphate (db-cAMP, membrane-permeable form of cAMP), the bell-shaped voltage dependence of cytosolic Ca 2+transients was dramatically broadened due to activation of SR Ca 2+ release at high membrane potentials (30-120 mV). . This is consistent with the spatial localization of the LCCs just above the RyRs across the junctional cleft (Franzini-Armstrong et al. 1999 (Leblanc & Hume, 1990;Lipp & Niggli, 1994;Kohomoto et al. 1994;Sipido, 1998). Additionally, there may be interplay between different Ca 2+ -entry pathways in initiation of SR Ca 2+ release. For example, it has been proposed that Ca 2+ entry through LCCs and reverse-mode NCX interact synergistically to activate RyR channels in the SR (Haworth et al. 1991;Litwin et al. 1998; Viatchenko-Karpinski & Györke, 2001). Integration of Ca 2+ -input signals from different entry pathways could provide an important mechanism for regulation of cardiac EC coupling and contractility. However, whether and how such integration of triggers for CICR occurs in cardiac muscle remain to be determined.Stimulation through the β-adrenergic pathway is a classical regulatory mechanism for controlling the contractile state of the myocardium. β-adrenergic agonists increase the amount of Ca 2+ released from the SR, resulting in the development of increased contractile force in cardiac muscle. The biochemical signalling mechanisms underlying β-adrenergic stimulation involve cAMP-dependent phosphorylation by protein kinase A (PKA) of certain target proteins including the LCC, RyR and phospholamban (Kranias et al. 1985;Shannon et al. 2001). In heart failure (HF), the Ca 2+ -releasing function of the SR appears to be compromised, resulting in depressed intracellular Ca 2+ transients and impaired contraction Eisner et al. 2003). Additionally the Ca 2+ -release machinery loses its ability to respond to adrenergic stimulation in myocytes from failing hearts, contributing to reduced contractile reserve in HF (Gomez et al. 1997;Pogwizd et al. 2001). The precise reasons for these HF-related changes in Ca 2+ transients and responsiveness to adrenergic stimulation are not known, and are currently the subject of intense debate.In the present study, we investigated the interplay between Ca 2+ entries via L-type Ca 2+ channels and reverse-mode NCX in activation of SR Ca 2+ release, focusing on the influence of adrenergic stimulation and changes occurring after induction of HF. Significantly, our results show that LCCs and NCX interact in a synergistic manner resulting in a larger total Ca 2+ entry than the arithmetic sum of entries mediated by each of these transport systems alone. This syne...
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