As part of a search for novel activators of Ca2+ pumping activity of cardiac SR- (sarcoplasmic reticulum), the EtOAc extract of the Jamaican sponge Plakortis halichondrioides was shown to be active. Bioassay-guided fractionation of the extract followed by preparative TLC and HPLC yielded several known and novel compounds. Three of the novel cyclic peroxides, plakortides F, G, and H (3, 4, and 5) are the subject of this report. Their structures including relative stereochemistry were established by interpretation of spectral data. Micromolar concentrations of plakortides F-H (3-5) were found to significantly enhance Ca2+ uptake by SR.
Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.
Cyclic ADP-ribose (cADPR), an endogenous metabolite of beta-NAD+, activates Ca2+ release from endoplasmic reticulum in sea urchin eggs via the ryanodine receptor (RyR) pathway. A similar role has been proposed in cardiac sarcoplasmic reticulum (SR), although this remains controversial. We therefore investigated the ability of cADPR to induce Ca2+ release from canine cardiac SR microsomes using fluo 3 to monitor extravesicular Ca2+ concentration. We found that cADPR induced Ca2+ release in a concentration-dependent manner, whereas neither its precursor, NAD+, nor its metabolite, ADP-ribose, elicited a consistent effect. In addition, an additive effect on calcium release between cADPR and 9-Me-7-Br-eudistomin-D (MBED), an activator of RyR, was found as well as no cross-desensitization between cADPR and MBED. Specific blockers of the RyR did not abolish the cADPR-induced Ca2+ release. These results provide evidence for cADPR-induced Ca2+ release from dog cardiac SR via a novel mechanism which is independent of RyR activation.
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