Specific inhibition of cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ pumps by H-89.

The isoquinolinesulfonamide H-89, an inhibitor of cyclic AMP-dependent protein kinases (EC 2.7.1.37, cAPrK), inhibited the Ca2+-ATPase activity of cardiac and skeletal muscle sarcoplasmic reticulum (SR) with concentrations giving half-maximal inhibition of 8.1 +/- 1.3 and 7.2 +/- 0.9 micromol/L, respectively. The effect of H-89 on cardiac SR Ca2+-ATPase (EC 3.6.1.38) was the same irrespective of the presence or absence of inhibitors of cAPrK and furthermore, was not affected by a neutralising monoclonal antibody raised against phospholamban. Thus, the action of H-89 in inhibiting SR Ca2+-ATPase would not appear to be mediated by inhibition of cAPrK to reduce the phosphorylation state of phospholamban. In both cardiac and skeletal muscle SR, the inhibition by H-89 was noncompetitive with respect to ATP at a low concentration of ATP (<1 mmol/L) and of a mixed pattern at high concentrations of ATP. H-89 produced a decrease in affinity of the SR Ca2+ pump to Ca2+ with an increase in the Km for Ca from 0.52 +/- 0.01 to 0.94 +/- 0.03 micromol/L (P < 0.05) in cardiac SR and from 0.39 +/- 0.01 to 0.79 +/- 0.02 micromol/L (P < 0.05) in skeletal muscle SR. These results suggest that H-89 inhibits SR Ca2+-ATPase by a direct action on the SR Ca2+ pump to decrease its affinity to Ca2+. Such an action may contribute to the pharmacological effect of H-89.

cADP-ribose releases Ca2+ from cardiac sarcoplasmic reticulum independently of ryanodine receptor.

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.

Mechanism of action of sarcoplasmic reticulum calcium-uptake activators--discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction.

The Ca2+ uptake by the sarcoplasmic reticulum (SR) can be affected by direct modulation of the Ca2+ pump or by removing the inhibitory effect of dephosphorylated phospholamban. The effect of these mechanisms was assessed using ellagic acid and 1-(3,4-dimethoxyphenyl)-3-dodecanone. Both compounds (30 micromol/l) enhanced SR-Ca2+ uptake in rabbit cardiomyocytes by 65.3 +/- 13% and 44.3 +/- 6.7% for 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid, respectively (at pCa 6.2). A similar effect was observed in cardiac SR microsomes (59.5 +/- 7.4% and 45.1 +/- 6.7) with 30 micromol/l 1-(3,4-dimethodoxyphenyl)-3-dodecanone and ellagic acid, respectively. 1-(3,4-Dimethoxyphenyl)-3-dodecanone increased Ca2+ storage by cardiac SR microsomes mainly at high [Ca2+] with a 57% increase of Vmax, whereas ellagic acid increased Vmax to a smaller extent (22%) and stimulated Ca2+ uptake at lower [Ca2+] with a leftward-shift of the pCa/ATPase relationship by pCa 0.24. Ellagic acid also differed from 1-(3,4-dimethoxylphenyl)-3-dodecanone in that it produced a Ca2+ sensitizing effect only in cardiac SR microsomes (by pCa 0.3) whereas 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulated the ATPase, at saturating Ca2+, in both cardiac and skeletal muscle SR vesicles. It is suggested that 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulates directly the Ca2+-ATPase activity, in contrast to ellagic acid which enhances the cardiac SR-Ca2+ uptake by interacting with phospholamban, as confirmed by the lack of additive effect between ellagic acid and monoclonal antibodies raised against phospholamban. 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid constitute attractive pharmacological tools to investigate the functional consequences of enhancing SR Ca2+, uptake by affecting different mechanisms.

Plakortides, novel cyclic peroxides from the sponge Plakortis halichondrioides: activators of cardiac SR-CA(2+)-pumping ATPase.

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.

Stereospecificity of myofibrillar calcium sensitivity and PDE inhibition in cardiotonic thiadiazinones.

In pyridazinone or thiadiazinone cardiotonic agents with one chiral centre, the PDE inhibitory action resides mainly in one enantiomer and the myofibrillar calcium sensitization mainly in the other. This phenomenon is observed when the chiral centre is located on the pyridazinone or thiadiazinone heterocycle, but cannot be extended to structures where the chiral centre is elsewhere on the molecule. For the first time a stereoselective synthesis of a 5-substituted 3,6-dihydro-6-methyl-2H-1,3,4-thiadiazine-2-one has been achieved and an absolute configuration is proposed.

Conformation of the cytoplasmic domain of phospholamban by NMR and CD.

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.

Effects of cyclopiazonic acid, an inhibitor of the Ca++ ATPase of sarcoplasmic reticulum, on Ca++ transport, contraction and relaxation in cardiac muscle.

Cyclopiazonic acid is a potent inhibitor of cardiac sarcoplasmic reticulum Ca++ ATPase. It scarely affects inotropism but significantly impairs lusitropism suggesting a greater role for cardiac sarcoplasmic reticulum in the control of cardiac relaxation than in the control of cardiac contraction.

A novel class of cardiotonic agents: synthesis and biological evaluation of 5-substituted 3,6-dihydrothiadiazin-2-ones with cyclic AMP phosphodiesterase inhibiting and myofibrillar calcium sensitizing properties.

As part of a search for new cardiotonic agents significantly sensitising the myocardial contractile proteins to calcium, together with cardiac cyclic AMP-PDE inhibitory activity, we have discovered that novel 5-substituted 3,6-dihydrothiadiazin-2-ones may fulfill both properties. The sensitising effect of the contractile proteins to calcium, assessed by the shift in the calcium sensitivity of canine cardiac myofibrillar magnesium-dependent ATPase, is determined by steric and electronic requirements. The requirements for phosphodiesterase inhibition, especially that of a near-planar arrangement for the phenyl and thiadiazin-2-one ring are consistent with those already described for analogous pyridazinones. The synthesis and structure-activity relationships are discussed.