Sibjotang Increases Atrial Natriuretic Peptide Secretion in Beating Rabbit Atria

Abstract

Sibjotang (Shizaotang), traditional herbal medicine formula, which was first documented in the Shanghanlun, has long been prescribed for the treatment of impairment of the body fluid homeostasis. The purpose of the present study was to identify the effects of Sibjotang on the secretion of a cardiac hormone, atrial natriuretic peptide (ANP), one of the main hormones involved in the regulation of the body fluid and blood pressure homeostasis. Water extract of Sibjotang increased ANP secretion concomitantly with an increase in atrial dynamics in a concentration-dependent manner. Sibjotang-induced increase in ANP secretion and positive inotropic effect were attenuated by GO6976 and LY333531, selective inhibitors of conventional protein kinase C, but not Rottlerin, an inhibitor of novel PKC δ . Similarly to the effect of Sibjotang, extracts of components of Sibjotang, Euphorbia kansui, and Daphne genkwa, but not Euphorbia pekinensis and Ziziphus jujuba, increased ANP secretion and atrial dynamics. Ingredients of Sibjotang, apigenin, rosmarinic acid, and salvianolic acid B decreased ANP secretion and atrial dynamics. These findings suggest that Sibjotang increases ANP secretion and atrial dynamics via activation of conventional protein kinase C signaling. This finding provides experimental evidence for the rationale in the use of Sibjotang in the treatment of impairment of the regulation of body fluid and blood pressure homeostasis.

Figure 1

Protocols for present experiments. Atria were paced 1.3 Hz. The values (means of two fractions) obtained before and after the addiction of vehicle (LY333531, GO6976, Rottlerin). Cont: control; SJ: Sibjotang; LY: LY333531; GO: GO697; Rott: Rottlerin; EK, Euphorbia kansui; EP: Euphorbia pekinensis; DG: Daphne genkwa; ZJ: Ziziphus jujube; ROS: rosmarinic acid; SAL: salvianolic acid B; API: apigenin. See Section 2.4 for details.

Figure 2

HPLC profile and H NMR analysis of Sibjotang. (a) Chemical structures of compounds. Number 1 is salvianolic acid B, number 2 is rosmarinic acid, number 3 is apigenin 7-O-b-glucuronide, number 4 is yuankanin, and number 5 is apigenin. It is analyzed by 1D- and 2D-NMR data; (b) HPLC chromatographic profile of the extract of SJ. Peak numbers correspond to structures given in (a), and the identity of the compounds was confirmed by retention times of isolated compounds.

Figure 3

Effects of Sibjotang (SJ) on secretory and contractile function in perfused beating rabbit atria. (a) Effects of vehicle (DMSO 0.1%) on ANP secretion (A), pulse pressure (B), and stroke volume (C). (b) Effects of SJ (100 μg/mL) on ANP secretion, pulse pressure, and stroke volume. (c) Effects of LY333531 (LY, 100 nM) on the SJ-induced increase in ANP secretion, pulse pressure, and stroke volume. Values are mean ± SE. Number of experiments: control, n = 8; SJ, n = 9; and LY + SJ, n = 8. ^∗∗∗ P < 0.001 versus mean values of two fractions before SJ.

Figure 4

Concentration-dependent effects of Sibjotang (SJ) on ANP secretion, pulse pressure, and stroke volume in atria. (a) Effects of SJ on ANP secretion. (b) Effects of SJ on pulse pressure. (c) Effects of SJ on stroke volume. Values are mean ± SE. Number of experiments: SJ (0 μg/mL, control), n = 6; SJ (60 μg/mL), n = 8; SJ (100 μg/mL), n = 8; SJ (300 μg/mL), n = 5; and SJ (1,000 μg/mL), n = 5. ^∗∗ P < 0.01, ^∗∗∗ P < 0.001 versus mean values of two fractions before SJ; ^# P < 0.05, ^### P < 0.001 versus control.

Figure 5

Recovery from the effects of Sibjotang (SJ) after replacing the buffer without the agent. (a) Recovery from the effects of SJ (1,000 μg/mL) on ANP secretion. (b) Recovery from the effects of SJ on pulse pressure. (c) Recovery from the effects of SJ on stroke volume. Values are mean ± SE. Number of experiments, n = 4. ^∗ P < 0.05, ^∗∗ P < 0.01 versus mean values of two fractions before SJ.

Figure 6

Effects of subtype-selective inhibitors of protein kinase C on the Sibjotang- (SJ-) induced increase in ANP secretion, pulse pressure, and stroke volume in atria. (a) Effects of inhibitors of protein kinase C on the SJ- (100 μg/mL) induced increase in ANP secretion. (b) Effects of inhibitors of protein kinase C on the SJ-induced increase in pulse pressure. (c) Effects of inhibitors of protein kinase C on the SJ-induced increase in stroke volume. Values are mean ± SE. Number of experiments: control, n = 8; SJ (100 μg/mL), n = 9; LY333531 (LY) 30 nM + SJ, n = 12; LY 30 nM + vehicle, n = 6; LY 100 nM + SJ, n = 8; LY 100 nM + vehicle, n = 5; GO6976 (GO) + SJ, n = 9; GO + vehicle, n = 5; Rottlerin (Rott) + SJ, n = 8; and Rott + vehicle, n = 6. ^∗ P < 0.05, ^∗∗∗ P < 0.001 versus SJ; ^## P < 0.01, ^### P < 0.001 versus control.

Figure 7

Effects of extract of component herbs of SJ, Euphorbia kansui (EK), Euphorbia pekinensis (EP), Daphne genkwa (DG), and Ziziphus jujube (ZJ) on ANP secretion, pulse pressure, and stroke volume in perfused beating rabbit atria. (a) Effects of extract of component herbs on ANP secretion. (b) Effects of extract of component herbs on pulse pressure. (c) Effects of extract of component herb on stroke volume. Values are mean ± SE. Number of experiments: control (C), n = 6; n = 7 for each of other groups. ^∗∗ P < 0.01, ^∗∗∗ P < 0.001 versus control.